A gene therapy for bacterial translocation in mice with whole body irradiation

Suzuki, Fujio

Abstract

In studies of severely burned mice, IL-10(-) IL-12(+) macrophages (M1 macrophages) distributed in lamina propria (LP) and mesenteric lymph nodes (MLN) have been identified as major effector cells in host antibacterial innate immunities against enterococcal translocation and subsequent sepsis. However, M1 macrophages are not generated easily when M2 macrophages are predominant. All 3 subsets of M2 macrophages (M2a, M2b and M2c macrophages) are inhibitory on resident macrophage conversion to M1 macrophages. Severely burned mice were subjected to gene therapy using CCL2 antisense oligodeoxynucleotides (ODN), because these mice early after burn injury were carriers of M2a and M2c macrophages. CCL2, a chemokine, converts resident macrophages to both M2a and M2c macrophages. In these mice, LP/MLN-M2a and M2c macrophages were changed to resident macrophages, and sepsis caused by enterococcal translocation is not developed in these mice subjected to CCL2 gene therapy. After oral infection with enterococci, bacterial translocation and subsequent sepsis were constantly demonstrated in mice irradiated with 3 to 7 Gy whole body gamma-rays (WBI-mice). These mice were shown to be carriers of M2b macrophages [LIGHT mRNA-expressing CCL1(+) IL-10(+) IL-12(-) macrophages]. CCL1 is a good target for the intervention of M2b macrophages, because this chemokine released from M2b macrophages themselves is essentially required for the maintenance of their properties. In our preliminary studies, LP/MLN-M2b macrophages isolated from WBI-mice were reverted to resident macrophages in cultures supplemented with CCL1 antisense ODN. In additional cultures with a bacterial antigen, resident macrophages reverted from LP/MLN-M2b macrophages were converted to M1 macrophages. From these facts, we have hypothesized that, in WBI-mice, the gene therapy using CCL1 antisense ODN is beneficial to improve host antibacterial innate immunities against gut-bacterial translocation and subsequent sepsis. To study this hypothesis, the following 2 Specific Aims are proposed: (i) to determine the effect of CCL1 antisense ODN on macrophage conversion from LP/MLN-M2b macrophages to resident macrophages in WBI-mice (resident macrophages will convert to M1 macrophages by bacterial antigen), and (ii) to control gut bacteria-associated sepsis by CCL1 antisense ODN in WBI-mice orally infected with enterococci. ELISPOT assay [IL-10(+) IL-12(-) F4/80 cells], quantitative RT-PCR (LIGHT mRNA, M2b F4/80 cells) and flow cytometry [IL-10(-) IL-12(+) or IL-10(+) IL-12(-) F4/80 cells] will be applied to detect various macrophage populations. The improved host resistance against enterococcal translocation and sepsis in mice 1 to 4 weeks after various severities of whole body gamma-irradiation will be measured by decreased bacterial growth in MLN, bacteremia and mortality rates. The strategy developed here will be possibly applied to various gut pathogens that are susceptible to host antibacterial innate immunities. Public Health Relevance: A new paradigm to treat gut bacteria-associated sepsis is urgent for persons who have radiation-associated damage in gastrointestinal system caused by terrorist acts or accidental exposure. Antibiotics are effective against these infections, however;antibiotic chemotherapies often encourage abnormal microflora and multi-antibiotic-resistant enterococci generation. The objective of this project is to develop a new gene therapy to mitigate sepsis stemming from bacterial translocation in mice irradiated with whole body gamma- rays.

Public Health Relevance

A new paradigm to treat gut bacteria-associated sepsis is urgent for persons who have radiation-associated damage in gastrointestinal system caused by terrorist acts or accidental exposure. Antibiotics are effective against these infections, however;antibiotic chemotherapies often encourage abnormal microflora and multi-antibiotic-resistant enterococci generation. The objective of this project is to develop a new gene therapy to mitigate sepsis stemming from bacterial translocation in mice irradiated with whole body gamma- rays.