Phage-Specific Diverse Effects of Bacterial Viruses on the Immune System

„With the increasing threat of antibiotic resistance, interest in phage therapy (PT) as a potential solution to this crisis has risen rapidly. Recently, several reports have been published describing successful treatment of patients with life-threatening antibiotic-resistant bacterial infections, including lung allograft recipients and treatment with genetically modified phages. Furthermore, the first PT center was opened in the USA, following the establishment of a similar unit in Belgium. These developments confirm our decision to establish the first such unit in 2005, operating in accordance with UKORE and national regulations, which has helped pave the way for future advances in PT as an option to combat the antibiotic resistance crisis. Extensive evidence from observational studies suggests the safety of PT. In addition, several clinical trials have been completed (including one conducted according to all required standards of good medical practice and evidence-based medicine) and are ongoing. However, these studies have yet to provide definitive proof of the efficacy of PT[1-4]. While the struggle for registration and market approval of phages as drugs continues, parallel data have accumulated suggesting that phages can interact not only with bacteria but also with eukaryotic cells (including cells of the immune system). Therefore, it cannot be ruled out that in the future, following phage discovery, research will shift toward phage-immune system interactions, whereas previously work on phage interactions with their natural target (bacteria) has dominated. It remains to be hoped that simultaneous advances in both research areas can bring positive results for human health, both in combating antibiotic-resistant bacterial infections and in developing new anti-inflammatory and immunomodulatory agents with minimal toxicity and satisfactory efficacy[4,5].

We have formulated a hypothesis stating that phages present in the gut can migrate into blood, lymph, and organs, mediate anti-inflammatory effects, and contribute to immunological tolerance and immune homeostasis—both in situ and at other sites in the body[6]. Study results confirm this, and furthermore, over 30 billion phages undergo transcytosis of the intestinal epithelium daily and distribute into blood, lymph, and organs[7]. In addition, other cell types, including immune cells, can also take up phages via the endocytic pathway[8].

The newly emerging concept of the phage, encompassing not only bacterial predators but also potential anti-inflammatory and immunomodulatory substances, requires detailed further investigation. A critical point that needs to be clarified is phage specificity in mediating certain immune responses. Phages are known for their high specificity toward bacteria, established for decades and used in phage typing to classify different bacterial strains. Are immunotropic activities also phage-specific, or do phages induce similar responses regardless of phage type?

It is believed that phage capsid proteins may be primarily responsible for the biological properties of the phage that are not related to interactions with bacteria. These proteins differ in their immunogenicity and can elicit different antibody responses to phages, which also depends on the route of administration. Furthermore, different strains of a homologous phage that recognize a particular bacterium can express different proteins[9,10] and confer different functions to the phage (e.g., persistence in circulation and antimetastatic effects). For example, a T4 phage mutant, HAP1, with a non-functional Hoc protein is more susceptible to liver Kupffer cells and is cleared more rapidly than its parental strain. There are also differences between HAP1 and T4 phages in their interactions with T cells and fibrinogen[11,12].
Initial studies on the effects of phages on other immune functions suggest that the effects may also vary depending on phage type. For example, purified T4 coliphage inhibits human T cell proliferation induced via the CD3-TCR complex, whereas purified staphylococcal phage exerts a co-stimulatory effect[12]. A detailed study of staphylococcal and Pseudomonas phages revealed that although these phages induced similar responses in human peripheral blood mononuclear cells through upregulation of gene expression of anti-inflammatory cytokines IL-1 receptor antagonist and suppressors of cytokine signaling 3, their influence on other immune functions was limited to the specific phage. A protolerogic and anti-inflammatory cytokine IL-10 was induced by all tested Pseudomonas phages but not by a staphylococcal phage. On the other hand, the latter phage caused TNFα, whereas only two of four tested Pseudomonas phages had similar effects. Furthermore, the TLR4 gene was downregulated exclusively by a Pseudomonas PMN phage, suggesting its anti-inflammatory effect (TLR4 activation causes secretion of proinflammatory cytokines)[13]. The diversity of phage effects on the immune system was also confirmed by recent data showing that a filamentous Pseudomonas Pf phage inhibits TNF production and phagocytosis, whereas Escherichia coli filamentous Fol phage has no such effects[8]. Moreover, our data suggest that both T4 coliphage and A5/80 Staphylococcus aureus phage significantly reduce the expression of human adenovirus genes, but viral DNA synthesis is inhibited only by T4 coliphage[14]. Furthermore, there is evidence that temperate and lytic phages may differ in their effects on the immune system[8]. Indeed, prophages are the main factor for bacterial immune system heterogeneity between strains, manifesting as variation in adaptive T and B cell immune responses of human lymphocytes in vitro to S. aureus and Streptococcus pyogenes[15].

Immunomodulatory and anti-inflammatory effects of phages may also be cell- and tissue-specific. Intranasal administration of 536_P1 (but not LM33-P1) coliphage in mice with experimental pneumonia resulted in an increase in antiviral lung cytokines and chemokines. Neither phage evoked changes in blood cytokine/chemokine levels, which also suggests that phage effects on the immune system may have different manifestations in different compartments of the body[16]. The ability of phages to mediate tissue-specific activity is confirmed by Pincus et al.[17], where staphylococcal phages did not induce proinflammatory cytokines in human peripheral blood mononuclear cells but were able to induce IFN-γ in human keratinocytes. Furthermore, we have shown that A5/80 staphylococcal phage increases IL-2 expression in the A549 cell line[18]; an activity not yet reported for phage effects on other cell types in in vitro studies. An increase in serum IL-2 levels in response to phage administration was also recently reported in mice treated with Acinetobacter baumannii phages, but their cellular source is unknown[19].

As mentioned, recent data suggest that phages can be internalized by mammalian cells and a large number undergo transcytosis via intestinal epithelial cells, while immune cells also internalize phages, particularly dendritic cells (DCs), monocytes, and B cells[7,8]. Recently, we described a distinct phage-dependent stimulation of the Hsp72 gene[18]. This induction of a known cellular chaperone may be a mechanism to protect cells undergoing transcytosis from potential injury by intracellular phages. Furthermore, Hsp72 is known to reduce T cell proliferation and cytokine secretion independently of the stimuli used and to inhibit DC ability to stimulate allogeneic T cells. This may suggest that Hsp72 could be used as an immunomodulator[20]. It has also been shown to suppress experimental arthritis in rats[21]. We have reported that phages can inhibit the development of collagen-induced arthritis in mice, an experimental model of rheumatoid arthritis[22]. Interestingly, Hsp72 has also been shown to suppress arthritis in this model[23]. It may well be that phage-dependent induction of Hsp72 is at least partially responsible for the inhibition of abnormal immune responses (including autoimmunity and hyperinflammation) caused by phages[24].
Phage interactions with immune cells may depend on specific phage receptors that enable these interactions. Currently, only limited data are available on the nature of such receptors. Pruzzo et al.[25] proposed that coliphages T3 and T7 could adhere to epithelial cells with their receptors for Klebsiella pneumoniae. Our hypothesis pointed to a Lys-Gly-Asp (KGD) sequence present in one of the capsid proteins of T4 phage as a potential ligand for cellular integrin receptors[24]. Lehti et al. showed that E. coli phage can recognize and bind neuroblastoma cells that display polysialic acid on their surface[26]. If polysialic acid is indeed a ligand for receptors of some phages, it could enable these phages to bind to immune cells, as the presence of polysialic acid has also been demonstrated on human DCs, NK cells, and a subpopulation of T cells[27,28]. Thus, it is likely that different phages can use different cellular ligands to bind and transcytose to target cells, including those of the immune system. In particular, even a single amino acid substitution in a phage capsid protein can result in a >1000-fold improvement in phage survival in mouse circulation, likely reflecting modified interactions between phages and phagocytes (and perhaps other cells endocytosing phages)[29].

Phages not only target specific bacteria but can also—at least partially—cause phage-specific immune responses. These findings open a new exciting field for further research on the significance of such responses for health and disease. Furthermore, these data suggest that a particular phage could be optimally selected for use in PT from different phage strains that recognize a particular bacterium, taking into account both its antibacterial activity and the type of immune response it can elicit. This is important in patients with immunodeficiencies, autoimmunity, allograft recipients, etc., who—depending on the nature of their disease—require immune stimulation or immunosuppression. Obviously, further research in this field can pave the way for the use of specific phages in immunomodulation.“

Translation of source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6802706/

Phage-specific diverse effects of bacterial viruses on the immune system
Andrzej Górski, Ryszard Międzybrodzki, Ewa Jończyk-Matysiak, Maciej Żaczek, and Jan Borysowski