Piotr Wierzbicki

Phage as a modulator of immune responses: practical implications for phage therapy.

Although the natural hosts for bacteriophages are bacteria, a growing body of data shows that phages can also interact with some populations of mammalian cells, especially with cells of the immune system. In general, these interactions include two main aspects. The first is the phage immunogenicity, that is, the capacity of phages to induce specific immune responses, in particular the generation of specific antibodies against phage antigens. The other aspect includes the immunomodulatory activity of phages, that is, the nonspecific effects of phages on different functions of major populations of immune cells involved in both innate and adaptive immune responses. These functions include, among others, phagocytosis and the respiratory burst of phagocytic cells, the production of cytokines, and the generation of antibodies against nonphage antigens. The aim of this chapter is to discuss the interactions between phages and cells of the immune system, along with their implications for phage therapy. These topics are presented based on the results of experimental studies and unique data on immunomodulatory effects found in patients with bacterial infections treated with phage preparations.

Phage as a Modulator of Immune Responses

Although the natural hosts for bacteriophages are bacteria, a growing body of data shows that phages can also interact with some populations of mammalian cells, especially with cells of the immune system. In general, these interactions include two main aspects. The first is the phage immunogenicity, that is, the capacity of phages to induce specific immune responses, in particular the generation of specific antibodies against phage antigens. The other aspect includes the immunomodulatory activity of phages, that is, the nonspecific effects of phages on different functions of major populations of immune cells involved in both innate and adaptive immune responses. These functions include, among others, phagocytosis and the respiratory burst of phagocytic cells, the production of cytokines, and the generation of antibodies against nonphage antigens. The aim of this chapter is to discuss the interactions between phages and cells of the immune system, along with their implications for phage therapy. These topics are presented based on the results of experimental studies and unique data on immunomodulatory effects found in patients with bacterial infections treated with phage preparations.

Rapamycin, unlike cyclosporine A, enhances suppressive functions of in vitro-induced CD4+CD25+ Tregs

BACKGROUND A growing body of data shows that CD4(+)CD25(+) regulatory T cells (Tregs) can induce transplantation tolerance by suppressing immune responses to allograft antigens. However, both the generation and the suppressive capacity of CD4(+)CD25(+) Tregs can be substantially affected by different immunosuppressive drugs used in clinical transplantation. The goal of this study was to compare the effects of cyclosporine A and rapamycin on the induction and suppressive functions of human CD4(+)CD25(+) Tregs in vitro. METHODS CD4(+)CD25(+) Tregs were induced in two-way mixed lymphocyte reaction (MLR) in the presence of rapamycin (Treg-Rapa) or cyclosporine A (Treg-CsA). Tregs were identified in MLR cultures by flow cytometry using anti-CD4, anti-CD25, anti-CTLA-4, anti-CD122, anti-GITR mAbs and ant-PE-FOXP3 staining sets. Suppressive capacity of induced Tregs was evaluated by their capability to inhibit anti-CD3 Ab-triggered proliferation of peripheral blood mononuclear cells (PBMCs), as measured by flow cytometry. The concentration of TGF-beta1 in culture supernatants was measured by enzyme-linked immunosorbent assay. RESULTS Although both rapamycin and cyclosporine A suppressed the induction of CD4(+)CD25(+) Tregs during MLRs, this effect was significantly more pronounced in cells cultured with cyclosporine. On the other hand, only rapamycin significantly decreased the percentage of CD4(+)CD25(+) Tregs which expressed GITR, a negative regulator of Tregs suppressive capacity. Importantly, Treg-Rapa, unlike Treg-CsA, displayed significant suppressive activity and were capable of inhibiting the proliferation of anti-CD3 Ab-activated PBMCs. This activity was likely mediated by TGF-beta1. CONCLUSIONS Rapamycin, unlike cyclosporine A, does not inhibit the function of CD4(+)CD25(+) Tregs. This implies that rapamycin could contribute to the development of transplantation tolerance by promoting the induction of functional CD4(+)CD25(+) Tregs. Moreover, our results suggest that rapamycin could be combined with functional Tregs.

Cladribine (2-chloro-deoxyadenosine, CDA): an inhibitor of human B and T cell activation in vitro.

We have previously shown that the novel immunosuppressive agent cladribine (CDA) inhibits human T and B cell lymphoproliferative responses and immunoglobulin synthesis in vitro, yet appears to be particularly efficacious as an inhibitor of B cell responses. We now report the effects of CDA on the human mixed lymphocyte reaction and on expression of T and B cell activation markers. CDA produced a significant inhibition of lymphocyte proliferation in human mixed lymphocyte reactions at a concentration of 10 nM. At concentrations of 10-100 nM the drug inhibited phytohaemagglutinin-induced expression of CD25 and HLA-D (by approximately 50%), but not phorbol myristate acetate-induced expression of CD69 on purified human T cells. At a concentration of 10 nM CDA totally abolished Staphylococcus aureus Cowan-induced expression of CD25 on purified B cells. These findings confirm that CDA is a potent immunosuppressive agent with some selectivity towards B cells. The drug may have potentially wide applications in clinical immunosuppression.

The Effects of T4 and A3/R Phage Preparations on Whole-Blood Monocyte and Neutrophil Respiratory Burst

Bacteriophages (viruses of bacteria) are currently considered a promising means of treating antibiotic-resistant infections. The main objective of this study was to evaluate the intensity of the whole-blood monocyte and neutrophil respiratory burst induced by purified preparations and lysates of the bacteriophages T4 and A3/R. While A3/R phage preparations did not induce a significant respiratory burst, T4 phage preparations increased the production of reactive oxygen species in a dose-dependent manner. However, the intensity of the phage-induced respiratory burst was much lower than that triggered by heat-inactivated Staphylococcus aureus cells. These results suggest that phage preparations are not likely to induce oxidative stress following their administration to patients.

A3R Phage and Staphylococcus aureus Lysate Do Not Induce Neutrophil Degranulation

The objective of this study was to evaluate the effects of A3R phage and Staphylococcus aureus lysate obtained after phage infection on neutrophil degranulation. The exocytosis of primary and secondary granules from neutrophils was investigated in vitro in whole blood specimens by flow cytometry based on the expression of specific markers of exocytosis (CD63 for primary granules and CD66b for secondary granules). We found that both A3R and S. aureus lysate had no significant effect on the exocytosis of primary and secondary granules. These data suggest that neither A3R virions nor any products of phage-induced lysis of S. aureus are likely to induce neutrophil degranulation in patients who are treated with phage preparations. Since neutrophil granules contain some potentially toxic proteins, our results provide an important argument for the safety of phage therapy. Moreover, these data indicate that the induction of neutrophil degranulation is not likely to contribute to antibacterial effects of phages.

LPS-Activated Monocytes Are Unresponsive to T4 Phage and T4-Generated Escherichia coli Lysate

A growing body of data shows that bacteriophages can interact with different kinds of immune cells. The objective of this study was to investigate whether T4 bacteriophage and T4-generated Escherichia coli lysate affect functions of monocytes, the key population of immune cells involved in antibacterial immunity. To that end, we evaluated how T4 and E. coli lysate influence the expression of main costimulatory molecules including CD40, CD80 and CD86, TLR2, TLR4 on monocytes, as well as the production of IL-6 and IL-12 in cultures of peripheral blood mononuclear cells (PBMCs). Separate experiments were performed on unactivated and LPS-activated PBMCs cultures. Both studied preparations significantly increased the percentage of CD14+CD16-CD40+ and CD14+CD16-CD80+ monocytes in unactivated PBMCs cultures, as well as the concentration of IL-6 and IL-12 in culture supernates. However, neither purified T4 nor E. coli lysate had any significant effect on monocytes in LPS-activated PBMCs cultures. We conclude that LPS-activated monocytes are unresponsive to phages and products of phage-induced lysis of bacteria. This study is highly relevant to phage therapy because it suggests that in patients with infections caused by Gram-negative bacteria the administration of phage preparations to patients and lysis of bacteria by phages are not likely to overly stimulate monocytes.

Human peripheral blood CD8+ CD28- T cells of renal allograft recipients do not express FOXP3 protein.

INTRODUCTION In recent studies, the FOXP3 molecule has been suggested to be a marker of a suppressor subset of human CD8+ CD28- T cells based on correlations between the level of its mRNA and allograft function. Because this transcriptional factor produces a protein, we suggest that these correlations should focus on the FOXP3 protein. The aim of our study was to evaluate whether FOXP3 protein was present in cells of the CD8+ CD28- population in the peripheral blood of renal allograft recipients and whether the level of CD8+ CD28- FOXP3+ cells correlated with allograft function. METHODS The study was performed on 30 renal allograft recipients with uneventful stable courses (n=18) or biopsy-proven chronic rejection (n=12). The immunosuppression was based on cyclosporine (n=12) or rapamycin (n=9). Peripheral blood mononuclear cells isolated from recipient blood samples were labeled with anti-CD8 and anti-CD28 MAbs conjugated with fluorochromes. After incubation, washing, and labeling using a PE anti-human FOXP3 Kit, we determined the percentage of cells by flow cytometry. RESULTS FOXP3 protein expression was not observed either in the CD8+ CD28- population, or the whole populations of CD8+ or CD28- cells among patient groups. CONCLUSIONS The expression of FOXP3 protein in CD8+ CD28- cells seems to be of a questionable value as a diagnostic tool for allograft function, it is probably not a marker for the CD8+ CD28- T cell subset.

Statins as immunomodulatory drugs

Statins (inhibitors of HMG-ConA reductase) are widely used as cholesterol-lowering agents and have beneficial action in patients with complications of atherosclerosis (coronary disease). In addition, recent data indicate that they can also modulate a variety of other biological processes inhibiting neointimal proliferation, reducing inflammation and stimulating osteogenesis (1 2 3). Moreover, our preliminary studies point to the role of statins as potential immunosuppressive agents (4). In this report, we confum and extend those initial observations.

Immunosuppression inhibits switch from naive to memory phenotype in human T lymphocytes

Recently, human T cells have been divided into memory (antigen-primed) and naive (unprimed) subsets, which vary in phenotype and function. Immunosuppressive drugs and heparin have been found to inhibit PHA-induced in vitro switching from naive to memory cell phenotype in a process that is at least partly independent of cell proliferation. Furthermore, T cells isolated from immunosuppressed renal allograft recipients were deficient in their in vitro PHA-induced transition from naive to memory phenotype.