Mari Kogiso

Lack of Th17 Cell Generation in Patients with Severe Burn Injuries

Immunodeficient patients with severe burn injuries are extremely susceptible to infection with Candida albicans. In addition to Th1 cells, IL-17–producing CD4+ T cells (Th17 cells) have recently been described as an important effector cell in host anti-Candida resistance. In this study, therefore, we tried to induce Th17 cells in cultures of severely burned patient PBMC by stimulation with the C. albicans Ag (CAg). In the results, the biomarkers for Th17 cells (IL-17 production and intracellular expression of IL-17 and retinoic acid receptor-related orphan receptor γt) were not displayed by burn patient PBMC stimulated with CAg, whereas these biomarkers of Th17 cells were detected in cultures of healthy donor PBMC stimulated with CAg. Burn patient sera were shown to be inhibitory on CAg-stimulated Th17 cell generation in healthy donor PBMC cultures; however, Th17 cells were induced by CAg in healthy donor PBMC cultures supplemented with burn patient sera that were previously treated with anti–IL-10 mAb. Also, the biomarkers of Th17 cells were not induced by CAg in healthy donor PBMC cultures supplemented with rIL-10. IL-10 was detected in serum specimens derived from severely burned patients. These results indicate that Th17 cells are not generated in burn patient PBMC cultures supplemented with CAg. IL-10, produced in response to burn injuries, is shown to be inhibitory on Th17 cell generation. The high susceptibility of severely burned patients to C. albicans infection might be influenced if burn-associated IL-10 production is intervened.

Propranolol as a modulator of M2b monocytes in severely burned patients

A role of immunosuppressive M2 monocytes (IL‐12–IL‐10+) on the increased susceptibility of severely burned patients to various opportunistic pathogens has been described. Among M2 monocyte subpopulations, M2b monocytes (IL‐17–CCL1+CXCL13–) are predominantly present in the peripheral blood of severely burned patients. In the present study, the rise and fall of M2b monocytes were examined in severely burned patients treated with propranolol. Catecholamine is known as an inducer of M2 monocytes, and propranolol is a competitive blocker of catecholamine binding to β‐adrenergic receptors. Twenty‐two children with 30% or more TBSA burn were enrolled in the study. Propranolol at a dose of 4 mg/kg/day was administered to these patients by feeding‐tube or mouth. Burn patient monocytes exhibited weak bactericidal activity. IL‐12 was produced by propranolol‐treated patient monocytes after stimulation with Staphylococcus aureus antigen, and the production of IL‐10, CCL1, CCL17, or CXCL13 by these monocytes was not demonstrated. These results indicate that a predominance of M2b monocytes in severely burned patients is intervened by the propranolol treatment. The increased susceptibility, to be associated with the appearance of M2b monocytes, of severely burned patients to opportunistic pathogens might be controlled by propranolol.

Effect of CCL2 antisense oligodeoxynucleotides on bacterial translocation and subsequent sepsis in severely burned mice orally infected with Enterococcus faecalis.

Severely burned mice are susceptible to sepsis stemming from Enterococcus faecalis translocation due to the impaired generation of M1 macrophages (M1Mϕs) in local translocation sites. In our previous studies, CCL2 has been characterized as a major effector molecule on the burn‐associated generation of M2Mϕs, an inhibitor cell type for resident Mϕ conversion into M1Mϕs. In this study, we tried to protect burned mice orally infected with E. faecalis utilizing CCL2 antisense oligodeoxynucleotides (ODNs). We show that M2Mϕs in mesenteric lymph nodes (MLNs) were not demonstrated in burned mice treated with CCL2 antisense ODNs. M1Mϕs were not induced by heat‐killed E. faecalis from resident Mϕs transwell‐cultured with mesenteric lymph node macrophages (MLN‐Mϕs) from burned mice, while M1Mϕs were induced by the same antigen from resident Mϕs transwell‐cultured with Mϕs which were isolated from burned mice treated with CCL2 antisense ODNs. Bacterial growth in MLNs was shown in burned mice orally infected with a lethal dose of E. faecalis. However, after the same infection, sepsis did not develop in burned mice treated with CCL2 antisense ODNs. These results indicate that bacterial translocation and subsequent sepsis are controlled in burned mice orally infected with a lethal dose of E. faecalis by gene therapy utilizing CCL2 antisense ODNs.

Effect of IL-10 antisense gene therapy in severely burned mice intradermally infected with MRSA

The effect of IL-10 antisense oligodeoxynucleotides (ODN) on the susceptibility of burned mice to intradermal (i.d.) infection of methicillin-resistant Staphylococcus aureus (MRSA) was studied. Abscesses formed and sepsis did not develop in normal mice infected i.d. with 10(8)CFU/mouse of MRSA. Similarly, sepsis caused by MRSA i.d. infection did not develop and abscesses formed in burned mice treated with IL-10 antisense ODN. However, all of the burned mice treated with scrambled ODN (control group) died by infectious complications stemming from MRSA i.d. infection, and an MRSA-abscess did not form in these mice. Macrophages (Mϕ) isolated from the infection site tissue of burned mice that were treated with IL-10 antisense ODN were identified as M1Mϕ, while Mϕ isolated from burned mice that were treated with scrambled ODN were shown to be M2Mϕ. MRSA-abscesses formed in burned mice inoculated with M1Mϕ, and these mice resisted a lethal dose of MRSA i.d. infection. However, an abscess did not form, and sepsis caused by MRSA i.d. infection developed in burned mice that were inoculated with M2Mϕ. These results indicate that severely burned mice treated with IL-10 antisense ODN are resistant against i.d. infection with MRSA. M1Mϕ appeared in the infection site tissues of severely burned mice that were treated with IL-10 antisense ODN may play a role on the abscess formation and inhibiting sepsis caused by MRSA i.d. infection.

Impaired ability of burn patient neutrophils to stimulate β-defensin production by keratinocytes.

Immunosuppressive neutrophils (PMN‐II) appearing in association with burn injury have a role on the increased susceptibility of burn patients to various infections. In the present study, the role of PMN‐II on the production of human β‐defensins (HBDs), important molecules on host antimicrobial innate immunities, by human keratinocytes was studied. Normal human epidermal keratinocytes (NHEKs) were cultured with neutrophils (PMNs) isolated from burn patients or healthy volunteers in dual‐chamber transwells. Culture fluids harvested 24 h after cultivation were assayed for HBDs using enzyme‐linked immunosorbent assay. Also, these culture fluids were assayed for their antimicrobial activities by a standard colony‐counting method using Pseudomonas aeruginosa. In the results, PMNs isolated from peripheral blood of burn patients were confirmed as PMN‐II, because these cells produced CC‐chemokine ligand 2 (CCL2), but not interleukin (IL)‐12 and CC‐chemokine ligand 3 (CCL3). Culture fluids of NHEK transwell‐cultured with healthy PMNs exhibited strong killing activities against P. aeruginosa (96% inhibition), however, the growth of bacteria was not dramatically inhibited by the culture fluids of NHEK transwell‐cultured with burn‐patient PMNs (36% inhibition). IL‐12 and CCL3 containing culture fluids of healthy PMNs stimulated with the bacterial antigen or rCCL3 and rIL‐12 enhanced the production of HBD2 and HBD3 by NHEK, whereas CCL2 containing culture fluids of burn‐patient PMN stimulated with the antigen or rCCL2 inhibited the HBD production by NHEK. These results indicate that PMN‐II appearing in association with burn injury contribute to the decreased production of HBDs in thermally injured patients.