Summary

Bacteriophages (viruses of bacteria) are used in the treatment of antibiotic‑resistant infections. Moreover, they are an important component of the mucosal microbiota. The objective of this study was to investigate the effects of T4 and A5/80 bacteriophages on the expression of genes involved in antimicrobial immunity, including Toll‑like receptors.

The expression of genes was determined in the A549 cell line using RT2 Profiler PCR Array.

Purified T4 and A5/80 phage preparations significantly affected the expression of 7 and 10 out of 84 examined genes, respectively.

Our results are important for phage therapy of bacterial infections and provide novel insights into the role of phages from the mucosal microbiota. They may also lead to novel applications of phages as antiviral and immunomodulatory agents.

Introduction

Bacteriophages (phages) are viruses of bacteria. The antibacterial activity of phages has been studied in great detail and has enabled their use in the treatment of bacterial infections, including those caused by antibiotic‑resistant strains [17]. Furthermore, bacteriophages present on mucous membranes may be involved in antibacterial immunity [5]. There are also reports showing that phages can exert other effects. For instance, our research revealed that phages can affect functions of some populations of immune cells [11]. Moreover, there are some data in the literature to suggest that phages, as well as some components of phage virions, may exert antiviral effects [27]. In this regard, our previous study showed that T4 bacteriophage can inhibit both the adsorption and replication of the adenovirus in the human lung carcinoma A549 cell line [30]. The main objective of the present study was to investigate whether phages can affect the expression of genes encoding proteins involved in antimicrobial immunity, including Toll‑like receptors (TLRs) and proteins mediating TLR signaling pathways [35]. We hypothesized that the antiviral effects of phages found in our previous study [30] might be mediated at least in part by the induction of these genes.

Material and methods

Purified phage preparations

Escherichia coli T4 phage [38] was obtained from the American Type Culture Collection (ATCC; USA). Staphylococcal A5/80 phage [25] was from the bacteriophage collection of the Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences (HIIET PAS). Bacterial strains for phage propagation (E. coli B and Staphylococcus aureus 80 for T4 and A5/80, respectively) were from the Polish Collection of Microorganisms at the HIIET PAS. Crude bacterial lysates of both phages were prepared according to the modified method of Ślopek et al. [33]. Purified preparations of T4 and A5/80 phages were prepared according to the modified protocol developed at the HIIET PAS [6, 26]. The lipopolisaccharide (LPS) level in purified phage preparations was determined using a QLC1000 Endpoint Chromogenic LAL test kit (Lonza, Switzerland) according to the manufacturer’s instructions. Phage titer was measured by the two‑layer method of Adams [2]. Stock preparations of both phages (1010 plaque forming units 
(PFU)/ml) were diluted with a culture medium before they were added to the cell culture.

Preparation of LPS from E. coli B

E. coli B culture was grown for 24 h at 37°C in liquid LB medium, pH 7.0 in 10L BioFlo415 fermenter (Eppendorf, Germany) as described by Kaszowska et al. [21]. LPS was isolated by the PCP (phenol/chloroform/petroleum) method [8] for the extraction of R lipopolysaccharides and purified by ultracentrifugation (105.000 
× g, 4 h, 4°C). It was then diluted with PBS and dispersed with the use of Sonopuls HD 2070 ultrasonic homogenizer connected to a UW 2070 HF‑generator (both from Bandelin, Germany). Its activity in the stock suspension measured in the LAL test, as described earlier, was 5000 EU/ml. It was dispersed again just before it was diluted with a culture medium and added to the cell culture. LPS suspension was used as an additional control for phage preparations.

Cell line and cell culture

The human lung carcinoma cell line A549 (ATCC CCL‑185) was obtained from LGC Promochem. A549 cells were grown in a culture medium composed of Eagle’s minimum essential medium (MEM) supplemented with 10% foetal bovine serum (FBS, Thermo Scientific) and antibiotic‑antimycotic solution (100 U/ml penicillin G, 100 mg/ml streptomycin and 0.25 µg/ml amphotericin B; Sigma‑Aldrich) and maintained at 37°C in a humidified atmosphere of 5% CO2 in air. Before experiments, cell suspensions were seeded in 96‑well microplates (Nunclon Surface, Nunc) at a density of 20.000 cells/200 µl per well and incubated for 24–36 h to form a confluent monolayer. Next, the cell culture medium was discarded and cells were treated for 2 h with one of the following additives at a volume of 200 µl: T4 preparation, A5/80 preparation, culture medium (untreated control), and E. coli B LPS at the final activity of 0.5 endotoxin units (EU)/ml, which corresponded to the final endotoxin activity in cultures incubated with phages (LPS control). The titers of both phages were adjusted so as to obtain the ratio of 100 phage particles per one cell. After incubation, the cell culture medium was discarded and cells were harvested for RNA isolation. Experiments were done in three independent replicates.

Evaluation of gene expression

RNA was isolated from A549 cells using an RNeasy Mini kit (Qiagen) according to the manufacturer’s instructions. RNA concentration and purity was determined with a NanoDrop spectrophotometer (Thermo Fisher Scientific). Expression of genes was determined with reverse‑transcripted real‑time PCR (RT‑qPCR) using Toll‑like receptor signaling pathway RT2 Profiler PCR Array Format F (Qiagen) by Roche LightCycler 480 according to the manufacturer’s protocol. This assay allows for the simultaneous evaluation of expression of 84 genes with the specificity and sensitivity of quantitative real‑time PCR. We determined the expression of gene encoding for Toll‑like receptors, proteins involved in pathogen‑specific responses, Toll‑like receptor signaling, downstream signaling of Toll‑like receptors, proteins involved in the regulation of adaptive immunity, Toll‑like receptor interacting proteins and adaptors and downstream effectors of Toll‑like receptor signaling. A complete list of these genes is available online as Supplementary Information. The level of genomic DNA contamination, the possibility of inhibition of the reverse‑transcription reaction, the presence of PCR amplification inhibitors, the cycling conditions, and the relative sensitivity of the instrument were validated according to the RT2 Profiler PCR Array manufacturer’s guide.

Data analysis

The relative transcript levels of genes of interest were analyzed using the ∆∆CT method according to the RT2 Profiler PCR Array manufacturer’s guide. The average CT of 5 housekeeping genes (ACTB, B2M, GAPDH, HPRT1, and RPLP0) was used for normalization. Difference in mRNA expression between T4, A5/80 or LPS treated groups and the untreated control was expressed as fold‑change (f‑c) values (2‑ΔΔCT). We considered it significant when there was at least two‑fold change: f‑c ≤0.5 or f‑c ≥2 (sample in triplicates) [8]. The range of f‑c was indicated by 2−(ΔΔC T + SD) and 2−(ΔΔCT − SD), where SD for ΔΔCT was calculated based on standard deviations of ΔCT values for treated and untreated groups [19]. To exclude potential bias due to averaging of data transformed through the 2−ΔΔCT equation, statistical analysis of ΔΔCT values obtained for each experiment was done with the use of a one‑sample t‑test with a 1.0 reference value [29]. The significance for this test was set at 5% (p‑value <0.05). Analysis was performed using SigmaPlot 12.3 software (Systat Software, Germany).

Results

The objective of this study was to investigate the effects of purified preparations of two bacteriophages – T4 and A5/80 – on the expression of genes encoding for proteins involved in the induction of antimicrobial immune responses. T4 is a classic E. coli phage that has been extensively characterized at the molecular and genetic level [38]. We used this phage because in our previous study it induced significant antiviral effects by blocking both the adsorption and replication of human adenovirus in the A549 cell line [30]. In addition, in the present study, to investigate whether antiviral effects can also be induced by other phages, we used staphylococcal A5/80 phage [25]. We hypothesized that antiviral effects of phages found in our previous study might be mediated (at least in part) by inducing some genes involved in antiviral immunity. To verify our hypothesis, we used an RT2 Profiler PCR Array, which allows for the simultaneous evaluation of expression of 84 genes, including those encoding individual TLRs and proteins involved in TLR signaling. This assay determines the expression of the examined genes with the sensitivity and specificity of quantitative real‑time PCR. Expression of genes in A549 cells was determined after 2‑h incubation with phage preparations. Compared with control cultures, T4 and A5/80 phage preparations significantly affected the expression of 7 and 10 genes, respectively (Table 1).

No.	Gene	Fold‑change value
		T4 vs control		A5/80 vs control		LPS vs control
1	CD80	1.4	(1.1‑1.9)	3.8	(2.8‑5.1) *	1.0	(0.9‑1.1)
2	CD86	3.2	(3.0‑3.4)***	1.3	(0.9‑1.9)	0.9	(0.8‑1.1)
3	CLEC4E	6.3	(6.0‑6.5)***	9.4	(7.58‑11.9)**	4.0	(1.1‑14.0)
4	HSPA1A	5163.0	(1667.1‑15990.0)***	7931.3	(2878.0‑21856.8)**	5895.2	(2145.5‑16198.6)**
5	IFNB1	2.3	(0.8‑7.0)	1.5	(0.8‑2.8)	1.0	(1.0‑1.0)
6	IL1A	0.1	(0.0‑0.4) *	1.2	(1.0‑1.5)	0.4	(0.2‑1.0)
7	IL1B	1.4	(0.7‑2.9)	1.5	(0.7‑3.1)	0.2	(0.1‑0.5)
8	IL2	1.3	(1.2‑1.3)**	13.9	(9.4‑20.4)**	1.3	(0.9‑2.0)
9	IL6	3.0	(0.3‑29.0)	3.0	(0.5‑17.5)	2.5	(0.4‑15.1)
10	IL10	2.2	(0.8‑6.1)	1.4	(0.9‑2.1)	0.9	(0.8‑1.1)
11	IL12A	0.5	(0.3‑0.7)*	0.5	(0.4‑0.7)**	0.7	(0.5‑1.0)
12	LTA	1.0	(0.2‑4.8)	5.1	(1.3‑19.9)	0.9	(0.2‑4.4)
13	TNF	7.0	(3.3‑14.9)*	2.3	(1.7‑3.1)*	4.4	(1.0‑19.7)
14	CSF2	1.3	(0.5‑3.3)	0.7	(0.4‑1.1) *	0.3	(0.2‑0.5)*
15	CSF3	0.7	(0.3‑1.5)	4.0	(2.7‑5.7)**	1.1	(0.3‑3.9)
16	TLR2	2.1	(0.9‑4.8)	1.3	(0.9‑1.9)	1.1	(0.8‑1.6)
17	TLR4	0.2	(0.0‑1.4)	0.4	(0.0‑3.5)	0.4	(0.2‑1.3)
18	TLR10	2.6	(0.8‑8.8)	2.8	(2.3‑3.5)*	3.8	(1.2‑11.8)
19	LY96	0.8	(0.6‑1.2)	0.4	(0.4‑0.5)**	0.2	(0.1‑1.1)
20	BTK	1.5	(0.9‑2.6)	3.8	(0.9‑16.4)	0.9	(0.7‑1.1)
21	REL	1.0	(0.7‑1.6)	2.1	(1.5‑2.8)	0.9	(0.7‑13)
22	FOS	0.4	(0.3‑0.7)	0.7	(0.6‑0.9)	0.6	(0.4‑0.9)

Table 1. Genes whose overexpression (fold change ≥ 2.0) or expression inhibition (fold change ≤ 0.5) in A549 cells was observed following at least one of individual treatments (T4 phage preparation, A5/80 phage preparation, or E. coli B LPS)

Legend: Shown are fold‑changes (and their range) compared with untreated control cultures (values representing at least two‑fold change are shown in bold). Significance of the difference in gene expression: *p <0.05; **p <0.01; ***p <0.001.

The most striking effect was high overexpression of the HSPA1 gene in cells treated both with T4 and A5/80 compared with control cultures (5163.0 f‑c and 7931.3 f‑c, respectively). However, this effect was probably mediated by residual LPS present in both preparations, because control LPS suspension increased the expression of this gene to a comparable extent (5895.2 f‑c).

Moreover, both T4 and A5/80 significantly increased the expression of the CLEC4E gene (6.3 f‑c and 9.4 f-c, respectively); the effect of LPS on this gene was weaker (4.0 f‑c; Table 1).

Of the 10 examined TLR genes (TLR1‑10), only the expression of TLR10 was significantly increased in A549 cells cultured with A5/80 phage preparation (2.8 f‑c).

Other genes whose expression in cells cultured with phage preparations was significantly affected were some cytokine and colony‑growth factor genes. Both phages influenced the expression of TNF and IL12A genes. Moreover, T4 affected the expression of the IL1A gene, while A5/80 changed the expression of the IL2, CSF2 and CSF3 genes.

Remarkably, a highly significant increase in IL2 gene expression was observed following treatment with A5/80 (13.9 f‑c; Table 1). Since LPS did not significantly affect the expression of any of these genes (except for CSF2), these changes were most likely caused by phage virions themselves.

Moreover, both phage preparations significantly affected the expression of a number of other genes. In particular, T4 preparation significantly increased the expression of the CD86 gene (3.2 f‑c compared with control cultures), while A5/80 preparation induced the CD80 gene (3.8 f‑c) and reduced the expression of the LY96 gene.

In addition, there were several genes whose overexpression (fold change ≥ 2.0) or expression inhibition (fold change ≤ 0.5) in A549 cells cultured with phage preparations was observed, but fell short of statistical significance; these genes are also listed in Tab. 1. These include TLR2, TLR4, IL6, IL10, IFNB1 and BTK.

Discussion

The most striking effect found in our study was a very high overexpression of the HSPA1 gene following treatment with either phage preparation. This gene encodes heat shock 70 kDa protein 1A (HSP1A), also called Hsp72, a major heat shock protein involved in a number of cellular activities, including protein synthesis, folding, and translocation into organelles as well as the assembly of multiprotein complexes. Generally, this protein is upregulated in response to cellular stress and it has cytoprotective functions [23]. However, it needs to be stressed that in other experiments neither of the studied phage preparations nor LPS exerted any cytotoxic effects on A549 cells [30]. The mechanism and potential consequences of the induction of the HSPA1A gene by phage preparations need to be investigated in further studies.

An interesting finding was a significant increase in TLR10 gene expression in cells treated with A5/80 phage preparation. TLR10 is unique among TLRs in that its activation can induce anti‑inflammatory effects [20]. Thus, by increasing the expression of this gene some phages might exert anti‑inflammatory activity. However, this needs to be verified by further studies. We also found that T4, but not A5/80 phage preparation, increased the expression of the TLR2 gene. This finding is of potential importance, because, according to a recent report, TLR2 is involved in the induction of initial antiviral immune response as a result of the recognition of the repeating protein subunit pattern common to many viral capsids [32]. Therefore, an increase in the expression of TLR2 may have mediated (at least in part) antiviral activity of T4 phage found in our previous study [30]; however, this needs to be verified by future studies.

Another important result is a reduction in the expression of the TLR4 gene induced by both phage preparations. TLR4 has been implicated in inflammatory reactions accompanying a number of diseases and pathological conditions, including sepsis. TLR4 signaling pathway modulators hold promise for use in sepsis [22]. Therefore, our finding may suggest a novel non‑bactericidal mechanism that could contribute to the therapeutic effects of phage preparations in sepsis. In fact, as discussed in our recent paper, there are solid data in the literature to support the initiation of clinical trials to evaluate the safety and the efficacy of phages in sepsis [12]. Furthermore, phage‑mediated interference with TLR signaling might promote beneficial therapeutic effects in autoimmune liver diseases, inflammatory bowel disease (IBD), allergies and graft‑versus‑host disease (GVHD) [13, 14, 15, 16].

Moreover, we found that both phage preparations significantly increased the expression of the CLC4E gene which encodes C‑Type Lectin Domain Family 4 Member E, also referred to as Macrophage‑inducible C‑type lectin and Mincle. Like TLRs, it is an important pattern‑recognition involved in the induction of antibacterial and antifungal immune responses [24]. Thus, it is possible that the induction of this gene might to some extent contribute to the antibacterial effects of phage preparations.

A5/80 significantly increased the expression of the IL2 gene. This effect was likely mediated by some component(s) of A5/80 virions, especially capsid proteins. This data is in accord with what other authors have also found, namely, that phages can stimulate IL‑2 production [7]. IL‑2 drives NK cell proliferation and production of lytic molecules; their cytolytic potential is reduced in lung cancer. Recent clinical trials on NK based therapies, including IL‑2 immunotherapy, showed promising results and fewer adverse effects in lung cancer and available data suggest that improving NK cell function may induce tumor regression [4]. Moreover, NK cells are important in immune defense against virus infections [36]. Moreover, NK cells have innate antibacterial activity against both intra‑ and extracellular pathogens and its augmentation may be considered to be a clinical intervention strategy [10]. Therefore, some experts believe that NK cells may become a tool to prevent or to combat bacterial infections [31]. Furthermore, NK cells play a vital role in IL-2‑ induced anticancer effects in A549‑bearing nude mouse model [39]. In this context, our data showing that A5/80 phage induces significant upregulation of the IL-2 gene in A549 may suggest the existence of additional mechanisms of IL-2 – dependent NK cell mediated immune protection against bacterial and viral infections and cancer triggered by some phages.

We also noted that T4 phage preparation increased the expression of gene encoding interlukin‑10 (IL‑10), the major anti‑inflammatory cytokine [40]. In our previous studies, we showed that during the treatment of bacterial infections phage preparations may exert anti‑inflammatory activity that is independent of a reduction in the number of pathogenic bacteria [28]. Thus, it is possible that anti‑inflammatory effects of some phage preparations might be mediated by an induction of IL‑10 (perhaps along with a reduction in the expression of TLR4 gene, as mentioned above). However, this hypothesis has to be verified by further experiments. Phage‑mediated induction of IL‑10 could open the way to phage application in treating some autoimmune diseases, inflammatory bowel disease, allergies and GVHD, where up‑regulation of IL‑10 production may promote beneficial therapeutic effects [13, 14, 15, 16]. Our results are in accord with the data of van Belleghem et al., [34] who showed that a Pseudomonas phage upregulates IL‑10 expression in human mononuclear cells, while a Staphyloccocus phage does not.

Other genes whose expression in A549 cells was changed following phage treatment include CD80, CD86 and BTK. All these genes encode proteins important for the immune system. CD80 and CD86 are proteins expressed largely by antigen presenting cells, which provide costimulatory signals essential for T cell activation [1]. The BTK gene encodes for Bruton’s tyrosine kinase, which plays an important role in B cell development, differentiation and signaling [37]. Potential biological effects resulting from the induction of these genes should be investigated in further studies.

A number of studies have shown that bacteriophages are an important component of the mucous microflora [3]. It appears that these phages may be involved in antimicrobial immunity [5]. Data obtained in the present study seem to support this. In particular, the induction of the TLR2 gene might account, at least in part, for antimicrobial effects of bacteriophages. The increase in the expression of IL‑10 might contribute to anti‑inflammatory activity of phages especially in the gut mucosa, which is in line with our earlier hypothesis that phages from the gut microbiota might contribute to the dampening of inflammatory reactions in the gut [18]. While our study is rather preliminary in nature, it provides a starting point for future investigations, which may generate new important data about the effects of phage preparations used for therapeutic purposes and phages being a natural component of the mucosal microbiota. Eventually, these studies may lead to novel applications of phage preparations as immunomodulatory and antiviral agents.

References

[1] Adams A.B., Ford M.L., Larsen C.P.: Costimulation blockade in autoimmunity and transplantation: The CD28 pathway. J. Immunol., 2016; 197: 2045–2050

[2] Adams M.H.: Bacteriophages, Interscience Publishers, Inc., New York 1959

[3] Aggarwala V., Liang G., Bushman F.D.: Viral communities of the human gut: metagenomic analysis of composition and dynamics. Mob. DNA, 2017; 8: 12

[4] Aktaş O.N., Öztürk A.B., Erman B., Erus S., Tanju S., Dilege Ş.: Role of natural killer cells in lung cancer. J. Cancer Res. Clin. Oncol., 2018; 144: 997–1003

[5] Barr J.J., Auro R., Furlan M., Whiteson K.L., Erb M.L., Pogliano J., Stotland A., Wolkowicz R., Cutting A.S., Doran K.S., Salamon P., Youle M., Rohwer F.: Bacteriophage adhering to mucus provide a non‑host‑derived immunity. Proc. Natl. Acad. Sci. USA, 2013; 110: 10771–10776

[6] Boratyński J., Syper D., Weber‑Dąbrowska B., Łusiak‑Szelachowska M., Poźniak G., Górski A.: Preparation of endotoxin‑free bacteriophages. Cell. Mol. Biol. Lett., 2004; 9: 253–259

[7] Cha K., Oh H.K., Jang J.Y., Jo Y., Kim W.K., Ha G.U., Ko K.S., Myung H.: Characterization of two novel bacteriophages infecting multidrug‑resistant (MDR) Acinetobacter baumannii and evaluation of their therapeutic efficacy in vivo. Front. Microbiol., 2018; 9: 696

[8] Dalman M.R., Deeter A., Nimishakavi G., Duan Z.H.: Fold change and p‑value cutoffs significantly alter microarray interpretations. BMC Bioinformatics, 2012; 13 (Suppl. 2): S11

[9] Galanos C., Lüderitz O., Westphal O.: A new method for the extraction of R lipopolysaccharides. Eur. J. Biochem., 1969; 9: 245–249

[10] Gonzales C.M., Williams C.B., Calderon V.E., Huante M.B., Moen S.T., Popov V.L., Baze W.B., Peterson J.W., Endsley J.J.: Antibacterial role for natural killer cells in host defense to Bacillus anthracis. Infect. Immun., 2012; 80: 234–242

[11] Górski A., Dąbrowska K., Międzybrodzki R., Weber‑Dąbrowska B., Łusiak‑Szelachowska M., Jończyk‑Matysiak E., Borysowski J.: Phages and immunomodulation. Future Microbiol., 2017; 12: 905–914

[12] Górski A., Jończyk‑Matysiak E., Łusiak‑Szelachowska M., Międzybrodzki R., Weber‑Dąbrowska B., Borysowski J.: The potential of phage therapy in sepsis. Front. Immunol., 2017; 8: 1783

[13] Górski A., Jończyk‑Matysiak E., Łusiak‑Szelachowska M., Międzybrodzki R., Weber‑Dąbrowska B., Borysowski J.: Bacteriophages targeting intestinal epithelial cells: a potential novel form of immunotherapy. Cell. Mol. Life Sci., 2018; 75: 589–595

[14] Górski A., Jończyk‑Matysiak E., Łusiak‑Szelachowska M., Międzybrodzki R., Weber‑Dąbrowska B., Borysowski J.: Phage therapy in allergic disorders? Exp. Biol. Med., 2018; 243; 534–537

[15] Górski A., Jończyk‑Matysiak E., Łusiak‑Szelachowska M., Weber‑Dąbrowska B., Międzybrodzki R., Borysowski J.: Therapeutic potential of phages in autoimmune liver diseases. Clin. Exp. Immunol., 2018; 192:1–6

[16] Górski A., Jończyk‑Matysiak E., Międzybrodzki R., Weber‑Dąbrowska B., Borysowski J.: “Phage transplantation in allotransplantation”: Possible treatment in graft‑versus‑host disease? Front. Immunol., 2018; 9: 941

[17] Górski A., Międzybrodzki R., Weber‑Dąbrowska B., Fortuna W., Letkiewicz S., Rogóż P., Jończyk‑Matysiak E., Dąbrowska K., Majewska J., Borysowski J.: Phage therapy: Combating infections with potential for evolving from merely a treatment for complications to targeting diseases. Front. Microbiol., 2016; 7: 1515

[18] Górski A., Weber‑Dąbrowska B.: The potential role of endogenous bacteriophages in controlling invading pathogens. Cell. Mol. Life Sci., 2005; 62: 511–519

[19] Greenhough J., Papadakis E.S., Cutress R.I., Townsend P.A., Oreffo R.O., Tare R.S.: Regulation of osteoblast development by Bcl‑2‑associated athanogene‑1 (BAG‑1). Sci. Rep., 2016; 6: 33504

[20] Hess N.J., Felicelli C., Grage J., Tapping R.I.: TLR10 suppresses the activation and differentiation of monocytes with effects on DC‑mediated adaptive immune responses. J. Leukoc. Biol., 2017; 101: 1245–1252

[21] Kaszowska M., Niedziela T., Maciejewska A., Lukasiewicz J., Jachymek W., Lugowski C.: Core oligosaccharide of Escherichia coli B‑the structure required for bacteriophage T4 recognition. Carbohydr. Res., 2015; 413: 51–54

[22] Kuzmich N.N., Sivak K.V., Chubarev V.N., Porozov Y.B., Savateeva‑Lyubimova T.N., Peri F.: TLR4 signaling pathway modulators as potential therapeutics in inflammation and sepsis. Vaccines, 2017; 5: E34

[23] Levada K., Guldiken N., Zhang X., Vella G., Mo F.R., James L.P., Haybaeck J., Kessler S.M., Kiemer A.K., Ott T., Hartmann D., Hüser N., Ziol M., Trautwein C., Strnad P.: Hsp72 protects against liver injury via attenuation of hepatocellular death, oxidative stress, and JNK signaling. J. Hepatol., 2018; 68: 996–1005

[24] Lu X., Nagata M., Yamasaki S.: Mincle: 20 years of a versatile sensor of insults. Int. Immunol., 2018; 30: 233–239

[25] Łobocka M., Hejnowicz M.S., Dąbrowski K., Gozdek A., Kosakowski J., Witkowska M., Ulatowska M.I., Weber‑Dabrowska B., Kwiatek M., Parasion S., Gawor J., Kosowska H., Głowacka A.: Genomics of staphylococcal Twort‑like phages – potential therapeutics of the post‑antibiotic era. Adv. Virus Res., 2012; 83: 143–216

[26] Łusiak‑Szelachowska M., Żaczek M., Weber‑Dąbrowska B., Międzybrodzki R., Kłak M., Fortuna W., Letkiewicz S., Rogóż P., Szufnarowski K., Jończyk‑Matysiak E., Owczarek B., Górski A.: Phage neutralization by sera of patients receiving phage therapy. Viral Immunol., 2014; 27: 295–304

[27] Międzybrodzki R., Fortuna W., Weber‑Dąbrowska B., Gorski A.: Bacterial viruses against viruses pathogenic for man? Virus Res., 2005; 110: 1–8

[28] Międzybrodzki R., Fortuna W., Weber‑Dąbrowska B., Górski A.: A retrospective analysis of changes in inflammatory markers in patients treated with bacterial viruses. Clin. Exp. Med., 2009; 9: 303–312

[29] Murie C., Woody O., Lee A.Y., Nadon R.: Comparison of small n statistical tests of differential expression applied to microarrays. BMC Bioinformatics, 2009; 10: 45

[30] Przybylski M., Borysowski J., Jakubowska‑Zahorska R., Weber‑Dąbrowska B., Górski A.: T4 bacteriophage‑mediated inhibition of adsorption and replication of human adenovirus in vitro. Future Microbiol., 2015; 10: 453–460

[31] Schmidt S., Ullrich E., Bochennek K., Zimmermann S.Y., Lehrnbecher T.: Role of natural killer cells in antibacterial immunity. Expert Rev. Hematol., 2016; 
9: 1119–1127

[32] Shepardson K.M., Schwarz B., Larson K., Morton R.V., Avera J., McCoy K., Caffrey A., Harmsen A., Douglas T., Rynda‑Apple A.: Induction of antiviral immune response through recognition of the repeating subunit pattern of viral capsids is toll‑like receptor 2 dependent. MBio, 2017; 8: e01356‑17

[33] Ślopek S., Durlakowa I., Weber‑Dąbrowska B., Kucharewicz‑Krukowska A., Dąbrowski M., Bisikiewicz R.: Results of bacteriophage treatment of suppurative bacterial infections. I. General evaluation of the results. Arch. Immunol. Ther. Exp., 1983; 31: 267–291

[34] Van Belleghem J.D., Clement F., Merabishvili M., Lavigne R., Vaneechoutte M.: Pro‑ and anti‑inflammatory responses of peripheral blood mononuclear cells induced by Staphylococcus aureus and Pseudomonas aeruginosa phages. Sci. Rep., 2017; 7: 8004

[35] Vijay K.: Toll‑like receptors in immunity and inflammatory diseases: Past, present, and future. Int. Immunopharmacol., 2018; 59: 319–412

[36] Waggoner S.N., Reighard S.D., Gyurova I.E., Cranert S.A., Mahl S.E., Karmele E.P., McNally J.P., Moran M.T., Brooks T.R., Yaqoob F., Rydyznski C.E.: Roles of natural killer cells in antiviral immunity. Curr. Opin. Virol., 2016; 16: 15–23

[37] Weber A.N., Bittner Z., Liu X., Dang T.M., Radsak M.P., Brunner C.: Bruton’s tyrosine kinase: An emerging key player in innate immunity. Front. Immunol., 2017; 8: 1454

[38] Yap M.L., Rossmann M.G.: Structure and function of bacteriophage T4. Future Microbiol., 2014; 9: 1319–1327

[39] Zhao L., Wang W.J., Zhang J.N., Zhang X.Y.: 5‑Fluorouracil and interleukin‑2 immunochemotherapy enhances immunogenicity of non‑small cell lung cancer A549 cells through upregulation of NKG2D ligands. Asian Pac. J. Cancer Prev., 2014; 15: 4039–4044

[40] Zhu L., Shi T., Zhong C., Wang Y., Chang M., Liu X.: IL‑10 and IL‑10 receptor mutations in very early onset inflammatory bowel disease. Gastroenterology Res., 2017; 10: 65–69

The authors have no potential conflicts of interest to declare.

References

• 1. Adams A.B., Ford M.L., Larsen C.P.: Costimulationblockade in autoimmunity and transplantation: TheCD28 pathway. J. Immunol., 2016; 197: 2045‑2050
  Google Scholar
• 2. Adams M.H.: Bacteriophages, Interscience Publishers,Inc., New York 1959
  Google Scholar
• 3. Aggarwala V., Liang G., Bushman F.D.: Viral communitiesof the human gut: metagenomic analysis of compositionand dynamics. Mob. DNA, 2017; 8: 12
  Google Scholar
• 4. Aktaş O.N., Öztürk A.B., Erman B., Erus S., Tanju S.,Dilege Ş.: Role of natural killer cells in lung cancer. J.Cancer Res. Clin. Oncol., 2018; 144: 997‑1003
  Google Scholar
• 5. Barr J.J., Auro R., Furlan M., Whiteson K.L., Erb M.L.,Pogliano J., Stotland A., Wolkowicz R., Cutting A.S., DoranK.S., Salamon P., Youle M., Rohwer F.: Bacteriophageadhering to mucus provide a non‑host‑derived immunity.Proc. Natl. Acad. Sci. USA, 2013; 110: 10771‑10776
  Google Scholar
• 6. Boratyński J., Syper D., Weber‑Dąbrowska B.,Łusiak‑Szelachowska M., Poźniak G., Górski A.: Preparationof endotoxin‑free bacteriophages. Cell. Mol. Biol.Lett., 2004; 9: 253‑259
  Google Scholar
• 7. Cha K., Oh H.K., Jang J.Y., Jo Y., Kim W.K., Ha G.U., KoK.S., Myung H.: Characterization of two novel bacteriophagesinfecting multidrug‑resistant (MDR) Acinetobacterbaumannii and evaluation of their therapeuticefficacy in vivo. Front. Microbiol., 2018; 9: 696
  Google Scholar
• 8. Dalman M.R., Deeter A., Nimishakavi G., Duan Z.H.:Fold change and p‑value cutoffs significantly altermicroarray interpretations. BMC Bioinformatics, 2012; 13 (Suppl. 2): S11
  Google Scholar
• 9. Galanos C., Lüderitz O., Westphal O.: A new methodfor the extraction of R lipopolysaccharides. Eur. J. Biochem.,1969; 9: 245‑249
  Google Scholar
• 10. Gonzales C.M., Williams C.B., Calderon V.E., HuanteM.B., Moen S.T., Popov V.L., Baze W.B., Peterson J.W.,Endsley J.J.: Antibacterial role for natural killer cells inhost defense to Bacillus anthracis. Infect. Immun., 2012;80: 234‑242
  Google Scholar
• 11. Górski A., Dąbrowska K., Międzybrodzki R.,Weber‑Dąbrowska B., Łusiak‑Szelachowska M.,Jończyk‑Matysiak E., Borysowski J.: Phages and immunomodulation.Future Microbiol., 2017; 12: 905‑914
  Google Scholar
• 12. Górski A., Jończyk‑Matysiak E., Łusiak‑SzelachowskaM., Międzybrodzki R., Weber‑Dąbrowska B., BorysowskiJ.: The potential of phage therapy in sepsis. Front. Immunol.,2017; 8: 1783
  Google Scholar
• 13. Górski A., Jończyk‑Matysiak E., Łusiak‑Szelachowska M., Międzybrodzki R., Weber‑Dąbrowska B., BorysowskiJ.: Bacteriophages targeting intestinal epithelial cells: apotential novel form of immunotherapy. Cell. Mol. LifeSci., 2018; 75: 589‑595
  Google Scholar
• 14. Górski A., Jończyk‑Matysiak E., Łusiak‑SzelachowskaM., Międzybrodzki R., Weber‑Dąbrowska B., BorysowskiJ.: Phage therapy in allergic disorders? Exp. Biol. Med.,2018; 243; 534‑537
  Google Scholar
• 15. Górski A., Jończyk‑Matysiak E., Łusiak‑SzelachowskaM., Weber‑Dąbrowska B., Międzybrodzki R., BorysowskiJ.: Therapeutic potential of phages in autoimmune liverdiseases. Clin. Exp. Immunol., 2018; 192: 1‑6
  Google Scholar
• 16. Górski A., Jończyk‑Matysiak E., Międzybrodzki R.,Weber‑Dąbrowska B., Borysowski J.: “Phage transplantationin allotransplantation”: Possible treatment ingraft‑versus‑host disease? Front. Immunol., 2018; 9: 941
  Google Scholar
• 17. Górski A., Międzybrodzki R., Weber‑Dąbrowska B.,Fortuna W., Letkiewicz S., Rogóż P., Jończyk‑Matysiak E.,Dąbrowska K., Majewska J., Borysowski J.: Phage therapy:Combating infections with potential for evolving frommerely a treatment for complications to targeting diseases.Front. Microbiol., 2016; 7: 1515
  Google Scholar
• 18. Górski A., Weber‑Dąbrowska B.: The potential roleof endogenous bacteriophages in controlling invadingpathogens. Cell. Mol. Life Sci., 2005; 62: 511‑519
  Google Scholar
• 19. Greenhough J., Papadakis E.S., Cutress R.I.,Townsend P.A., Oreffo R.O., Tare R.S.: Regulation of osteoblastdevelopment by Bcl‑2‑associated athanogene‑1(BAG‑1). Sci. Rep., 2016; 6: 33504
  Google Scholar
• 20. Hess N.J., Felicelli C., Grage J., Tapping R.I.: TLR10suppresses the activation and differentiation of monocyteswith effects on DC‑mediated adaptive immuneresponses. J. Leukoc. Biol., 2017; 101: 1245‑1252
  Google Scholar
• 21. Kaszowska M., Niedziela T., Maciejewska A., LukasiewiczJ., Jachymek W., Lugowski C.: Core oligosaccharideof Escherichia coli B‑the structure required for bacteriophageT4 recognition. Carbohydr. Res., 2015; 413: 51‑54
  Google Scholar
• 22. Kuzmich N.N., Sivak K.V., Chubarev V.N., PorozovY.B., Savateeva‑Lyubimova T.N., Peri F.: TLR4 signalingpathway modulators as potential therapeutics in inflammationand sepsis. Vaccines, 2017; 5: E34
  Google Scholar
• 23. Levada K., Guldiken N., Zhang X., Vella G., Mo F.R.,James L.P., Haybaeck J., Kessler S.M., Kiemer A.K., Ott T.,Hartmann D., Hüser N., Ziol M., Trautwein C., Strnad P.:Hsp72 protects against liver injury via attenuation ofhepatocellular death, oxidative stress, and JNK signaling.J. Hepatol., 2018; 68: 996‑1005
  Google Scholar
• 24. Lu X., Nagata M., Yamasaki S.: Mincle: 20 years ofa versatile sensor of insults. Int. Immunol., 2018; 30:233‑239
  Google Scholar
• 25. Łobocka M., Hejnowicz M.S., Dąbrowski K., GozdekA., Kosakowski J., Witkowska M., Ulatowska M.I.,Weber‑Dabrowska B., Kwiatek M., Parasion S., GaworJ., Kosowska H., Głowacka A.: Genomics of staphylococcalTwort‑like phages – potential therapeutics of thepost‑antibiotic era. Adv. Virus Res., 2012; 83: 143‑216
  Google Scholar
• 26. Łusiak‑Szelachowska M., Żaczek M.,Weber‑Dąbrowska B., Międzybrodzki R., Kłak M., FortunaW., Letkiewicz S., Rogóż P., Szufnarowski K.,Jończyk‑Matysiak E., Owczarek B., Górski A.: Phage neutralizationby sera of patients receiving phage therapy.Viral Immunol., 2014; 27: 295‑304
  Google Scholar
• 27. Międzybrodzki R., Fortuna W., Weber‑Dąbrowska B.,Gorski A.: Bacterial viruses against viruses pathogenicfor man? Virus Res., 2005; 110: 1‑8
  Google Scholar
• 28. Międzybrodzki R., Fortuna W., Weber‑DąbrowskaB., Górski A.: A retrospective analysis of changes ininflammatory markers in patients treated with bacterialviruses. Clin. Exp. Med., 2009; 9: 303‑312
  Google Scholar
• 29. Murie C., Woody O., Lee A.Y., Nadon R.: Comparisonof small n statistical tests of differential expressionapplied to microarrays. BMC Bioinformatics, 2009; 10: 45
  Google Scholar
• 30. Przybylski M., Borysowski J., Jakubowska‑ZahorskaR., Weber‑Dąbrowska B., Górski A.: T4 bacteriophage‑mediatedinhibition of adsorption and replicationof human adenovirus in vitro. Future Microbiol., 2015;10: 453‑460
  Google Scholar
• 31. Schmidt S., Ullrich E., Bochennek K., ZimmermannS.Y., Lehrnbecher T.: Role of natural killer cells in antibacterialimmunity. Expert Rev. Hematol., 2016; 9:1119‑1127
  Google Scholar
• 32. Shepardson K.M., Schwarz B., Larson K., MortonR.V., Avera J., McCoy K., Caffrey A., Harmsen A., DouglasT., Rynda‑Apple A.: Induction of antiviral immuneresponse through recognition of the repeating subunitpattern of viral capsids is toll‑like receptor 2 dependent.MBio, 2017; 8: e01356‑17
  Google Scholar
• 33. Ślopek S., Durlakowa I., Weber‑Dąbrowska B.,Kucharewicz‑Krukowska A., Dąbrowski M., BisikiewiczR.: Results of bacteriophage treatment of suppurativebacterial infections. I. General evaluation of the results.Arch. Immunol. Ther. Exp., 1983; 31: 267‑291
  Google Scholar
• 34. Van Belleghem J.D., Clement F., Merabishvili M.,Lavigne R., Vaneechoutte M.: Pro‑ and anti‑inflammatoryresponses of peripheral blood mononuclear cellsinduced by Staphylococcus aureus and Pseudomonas aeruginosaphages. Sci. Rep., 2017; 7: 8004
  Google Scholar
• 35. Vijay K.: Toll‑like receptors in immunity and inflammatorydiseases: Past, present, and future. Int. Immunopharmacol.,2018; 59: 391‑412
  Google Scholar
• 36. Waggoner S.N., Reighard S.D., Gyurova I.E., CranertS.A., Mahl S.E., Karmele E.P., McNally J.P., Moran M.T.,Brooks T.R., Yaqoob F., Rydyznski C.E.: Roles of naturalkiller cells in antiviral immunity. Curr. Opin. Virol., 2016;16: 15‑23
  Google Scholar
• 37. Weber A.N., Bittner Z., Liu X., Dang T.M., RadsakM.P., Brunner C.: Bruton’s tyrosine kinase: An emergingkey player in innate immunity. Front. Immunol., 2017;8: 1454
  Google Scholar
• 38. Yap M.L., Rossmann M.G.: Structure and function ofbacteriophage T4. Future Microbiol., 2014; 9: 1319‑1327
  Google Scholar
• 39. Zhao L., Wang W.J., Zhang J.N., Zhang X.Y.: 5‑Fluorouraciland interleukin‑2 immunochemotherapyenhances immunogenicity of non‑small cell lung cancerA549 cells through upregulation of NKG2D ligands. AsianPac. J. Cancer Prev., 2014; 15: 4039‑4044
  Google Scholar
• 40. Zhu L., Shi T., Zhong C., Wang Y., Chang M., Liu X.:IL‑10 and IL‑10 receptor mutations in very early onsetinflammatory bowel disease. Gastroenterology Res.,2017; 10: 65‑69
  Google Scholar

Full text