Fujio Suzuki

CCL17 and IL-10 as Effectors That Enable Alternatively Activated Macrophages to Inhibit the Generation of Classically Activated Macrophages

Classically activated macrophages (CAMφ) have been described as a major effector cell on the host’s innate immunities. However, CAMφ are not generated in immunocompromised hosts whose alternatively activated macrophages (AAMφ) predominate. In this study, the mechanism by which AAMφ suppress the ability of resident macrophages (RMφ) to generate CAMφ was investigated. AAMφ were isolated from peritoneal exudates of mice 2 days after third-degree thermal injuries affecting 15% total body surface area. CAMφ were generated from RMφ (peritoneal Mφ from normal mice) through stimulation with CpG DNA, a typical CAMφ inducer. RMφ did not polarize to CAMφ when they were cultured with AAMφ in a dual-chamber Transwell even when supplemented with CpG DNA. In addition, RMφ stimulated with CpG DNA did not convert to CAMφ when they were cultured with the culture fluids of AAMφ (AAMφ Culture-Sup). AAMφ Culture-Sup contained IL-6, IL-10, CCL17, PGE2, and TGF-β. Among these, CCL17 and IL-10 inhibited CAMφ generation. The ability of AAMφ Culture-Sup to inhibit CAMφ generation was eliminated when the Culture-Sup was treated with a mixture of mAbs directed against CCL17 and IL-10. These results indicate that CCL17 and IL-10 released from AAMφ inhibit CAMφ generation from RMφ stimulated with CpG DNA.

M2b Monocytes Predominated in Peripheral Blood of Severely Burned Patients

Severely burned patients were shown to be carriers of M2 monocytes, and all of the monocytes isolated from peripheral blood of severely burned patients (19 of 19 patients) were demonstrated as M2b monocytes (IL-12−IL-10+CCL1+ monocytes). Low levels of M2a (IL-12−IL-10+CCL17+ monocytes) and M2c monocytes (IL-12−IL-10+CXCL13+ monocytes) were demonstrated in peripheral blood of severely burned patients (M2a, 2 of 19 patients; M2c, 5 of 19 patients). M2b, M2a, and M2c monocytes were not detected in peripheral blood of healthy donors. However, M2b monocytes appeared when healthy donor monocytes were cultured in media supplemented with burn patient serum (15%). CCL2 was detected in sera of all burn patients, and M2b monocytes were not generated from healthy donor monocytes cultured with media containing 15% burn patient sera that were previously treated with anti-CCL2 mAb. In addition, M2b monocytes were generated from healthy donor monocytes in cultures supplemented with rCCL2. These results indicate that M2b monocytes are predominant in peripheral blood of severely burned patients who are carriers of CCL2 that functions to stimulate monocyte conversion from resident monocytes to M2b monocytes.

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.

Cryptococcal encephalitis in thermally injured mice is accelerated by type 2 T-cell responses.

Objective To explore the pathogenic role of burn-associated type 2 T-cell responses on the development of cryptococcal encephalitis in mice with severe thermal injuries. Design Experimental Cryptococcus neoformans infection in normal mice was compared with that in thermally injured mice (TI mice), normal mice treated with a mixture of interleukin (IL)-4 and IL-10, or normal mice inoculated with burn-associated type 2 T cells. Setting University research laboratory. Subjects Male BALB/c mice, 8 to 10 wks of age. Interventions We prepared four groups of mice as follows: a) normal mice, b) TI mice, c) normal mice treated with the IL-4/IL-10 mixture, and d) normal mice inoculated with burn-associated type 2 T cells. These groups of mice were anesthetized and exposed to 1 × 10 cells/mouse of C. neoformans intratracheally. Cryptococcal growth in brains and lungs in normal mice were compared with those of the other three groups. Also, cytokine-producing profiles of T lymphocytes from brains of both normal mice and TI mice were determined. Measurements and Main Results Compared with normal mice, TI mice were susceptible to C. neoformans infection. At the maximum (15 days after infection), numbers of C. neoformans organisms in brains of TI mice were 10 times higher than those of the pathogen in brains of normal mice. After stimulation with anti-CD3 monoclonal antibody, IL-4 (but not interferon gamma) was produced in cultures of T lymphocytes from brains of TI mice 15 days after the infection, whereas the same cell preparation from normal mice produced interferon gamma (but not IL-4). TI mice and mice that were treated with a IL-4/IL-10 mixture or inoculated with burn-associated type 2 T cells were equally susceptible to the cryptococcal infection. Conclusions Burn-associated type 2 T cells or their cytokine products play a key role in the severity of cryptococcal encephalitis that develops in TI mice.

Orosomucoid 1 drives opportunistic infections through the polarization of monocytes to the M2b phenotype.

Orosomucoid (ORM, composed of two isoforms, ORM1 and ORM2) has been described as an inducer of M2 macrophages, which are cells that decrease host antibacterial innate immunities. However, it is unknown which phenotypes of M2 macrophages are induced by ORM. In this study, healthy donor monocytes stimulated with ORM (ORM-monocytes) were characterized phenotypically and biologically. CCL1 (a biomarker of M2b macrophages) and IL-10 were detected in monocyte cultures supplemented with ORM1; however, CCL17 (a biomarker of M2a macrophages) and CXCL13 (a biomarker of M2c macrophages) were not produced in these cultures. All of these soluble factors were not detected in the culture fluids of monocytes stimulated with ORM2. Monocytes stimulated with ORM1 were characterized as CD64(-)CD209(-)CD163(+)CCL1(+) cells. MRSA and Enterococcus faecalis infections were accelerated in chimeras (NOD/scid IL-2Rγ(null) mice reconstituted with white blood cells) after inoculation with monocytes stimulated with ORM1 or treatment with ORM1; however, the infections were greatly mitigated in both chimeras inoculated with ORM1-stimulated monocytes and treated with ORM1, after an additional treatment with an inhibitor of M2b macrophages (CCL1 antisense ODN). These results indicate that ORM1 stimulates quiescent monocytes to polarize to M2b monocytes. The regulation of M2b macrophages may be beneficial in controlling opportunistic infections in patients with a large amount of plasma ORM1.

Lineage−CD34+CD31+ Cells That Appear in Association with Severe Burn Injury Are Inhibitory on the Production of Antimicrobial Peptides by Epidermal Keratinocytes

Antimicrobial peptides are major host defense effectors against Pseudomonas aeruginosa skin infections. Due to the lack of such peptide production, severely burned hosts are greatly susceptible to P. aeruginosa burn wound infection. β-Defensin (HBD) production by normal human epidermal keratinocytes (NHEK) was inhibited by lineage−CD34+ cells isolated from peripheral blood of severely burned patients. Lineage−CD34+ cells obtained from severely burned patients were characterized as CD31+, while healthy donor lineage−CD34+ cells were shown to be CD31− cells. Lineage−CD34+CD31− cells did not show any inhibitory activities on HBD-1 production by NHEK. CCL2 and IL-10 released from lineage−CD34+CD31+ cells were shown to be inhibitory on the peptide production by NHEK, while these soluble factors were not produced by lineage−CD34+CD31− cells. After treatment with a mixture of mAbs for CCL2 and IL-10, the culture fluids of lineage−CD34+CD31+ cells did not show any inhibitory activities on HBD-1 production by NHEK. Lineage−CD34+CD31+ cells that appear in association with burn injuries play a role on the inhibition of antimicrobial peptide production by skin keratinocytes through the production of CCL2 and IL-10.

Host Defense Antibacterial Effector Cells Influenced by Massive Burns

Abstract Infections in severely burned patients frequently occur as opportunistic infections. Opportunistic invading microbes are recognized by the hosts immune systems both to elicit immediate defense and to generate long-lasting adaptive immunity. The innate immune system is the first line of host defense against microbial invasion. Cells and tissues influenced by burn injury stimulate many cells related to the innate immune system. Burn-associated hyperinflammatory responses are induced during these host reactions. This immune perturbation leads to the subsequent development of extended immunosuppression caused by type 2 immune responses. To understand how and why host antibacterial defenses are suppressed by massive burns, this chapter discusses cell populations and their functions as involved in innate immunities influenced by severe burn injuries.

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 24u2009h 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.