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Yoshitaka Hirose, Shinji Murosaki, Yoshihiro Yamamoto, Yasunobu Yoshikai, Tomomi Tsuru, Daily Intake of Heat-Killed Lactobacillus plantarum L-137 Augments Acquired Immunity in Healthy Adults, The Journal of Nutrition, Volume 136, Issue 12, December 2006, Pages 3069–3073, https://doi.org/10.1093/jn/136.12.3069
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Abstract
Heat-killed Lactobacillus plantarum strain L-137 (HK-LP) is a potent inducer of IL-12 in vitro as well as in vivo in mice. HK-LP has been shown to suppress IgE production against food allergens, as well as tumor growth in mice, through IL-12 production, which induces the T helper (Th) 1 type immune response. To determine whether the intake of HK-LP influences immune function and the quality of life (QOL), a randomized, double-blind, placebo-controlled, parallel study was conducted in healthy subjects. Sixty subjects (30 men and 30 women, mean age 56.3 y) were randomly assigned to receive a capsule containing 10 mg of HK-LP daily or a matching capsule for 12 wk. Biomarkers for innate immunity such as the natural killer activity of peripheral blood mononuclear cells, neutrophil phagocytosis, and cell surface expression of CD64 on monocytes were measured every 4 wk. Biomarkers for acquired immunity such as concanavalin A (Con A)-induced proliferation, percentages of INF-γ and IL-4–producing cluster of differentiation (CD)4+ T cells (Th1:Th2 ratio), and the serum IgG4:IgG ratio were measured every 4 wk or at wk 0 and wk 12. Health-related QOL was assessed using a self-rating questionnaire with 26 items. Among the measured biomarkers, the percent change in Con A-induced proliferation and the Th1:Th2 ratio in the HK-LP group was greater than those in the control group (P = 0.036 and P = 0.002, respectively). The degree of improvement in QOL was higher in the HK-LP group than in the control group at wk 8 (P = 0.049) and tended to be higher at wk 12 (P = 0.092). These results suggest that a daily intake of HK-LP augments acquired immunity, especially Th1-related immune functions in healthy subjects, thereby improving the health-related QOL.
Introduction
Probiotics have been defined as live microbial food components that are beneficial for human health. Recently, because they exhibited beneficial effects equal to those of live microbes, genetically engineered microbes and nonviable microbes have been regarded as probiotics (1,2). Lactic acid bacteria, one of the most common types of probiotic bacteria, have been reported to exhibit beneficial effects on host homeostasis, including activation of the immune function (3,4). We showed previously that heat-killed Lactobacillus plantarum L-137 (HK-LP),5 a strain isolated from fermented food, is a potent inducer of IL-12 in vitro as well as in vivo in mice (5). Administration of HK-LP suppressed IgE production against naturally fed antigen in a mouse model of food allergy (5). HK-LP also inhibited tumor growth in mice transplanted with syngeneic tumor cells (6). These effects of HK-LP have been shown to be exerted through the induction of IL-12, which leads to a T helper (Th) 1 type immune response (5,6). Another study has also shown the effectiveness of lactobacilli on the Th1 response, in which activation of human dendritic cells by lactobacilli skews T cells toward Th1 polarization (7). Augmentation of the Th1 response may be beneficial for individuals in Westernized societies, because the better public hygiene and fewer infections in these societies may reduce the Th1 response, thereby increasing the risk of developing allergies (8).
Various lines of evidence in human studies have revealed that foods and their components can improve immune function in a wide range of human subjects whose immune function is poorer than expected. Systematically analyzing such evidence, Kaminogawa et al. (9) have concluded that the improvement of immune function by food ingredients can be evaluated by measuring the general biomarkers for innate immunity [phagocytic activity and natural killer (NK) activity] and acquired immunity (delayed-type hypersensitivity, antigen-specific antibody production, proliferative response of T cells, and T cell number). Moreover, improvement of these biomarkers has been shown to correlate with reduction of the incidence of pathogenic infection (9).
Poor immune function likely results not only in high susceptibility to infections but also deterioration of the health-related quality of life (QOL). In fact, among patients with chronic fatigue syndrome, individuals with lower T cell proliferation have been reported to show poor health-related QOL scores (10). In addition, we have previously reported that nigerooligosaccharide, which is an immunopotentiating agent, augments T-cell mitogen response in association with improvement of the health-related QOL in the elderly (11). Therefore, enhancement of immune function may lead to improvement of the health-related QOL.
The objective of this study was to examine whether intake of HK-LP affects innate and acquired immune functions and health-related QOL in healthy subjects.
Subjects and Methods
Subjects.
Sixty healthy subjects (mean age 56.3 y) participated in this study. They comprised 36 middle-aged (40–64 y, 18 men and 18 women) and 24 elderly subjects (>64 y, 12 men and 12 women). The eligibility of the subjects was assessed by interviewing them and consulting with their health care providers. The inclusion criteria were general good health and a willingness to follow the trial guidelines. Exclusion criteria were any history of any illness, including allergic disease, currently being treated with a prescribed medicine, and intolerance of soy and milk products. Eating habits were not considered for the criteria, but the subjects were instructed to continue with their habitual diets during the study. Approval of the protocol was obtained from the Institutional Review Board of Medical Co. LTA (Fukuoka, Japan) and the study was conducted in accordance with the Declaration of Helsinki. The procedures had been fully explained to the subjects and written informed consent was obtained from each subject before the beginning of the study.
Preparation of HK-LP.
LP20 (House Wellness Foods), which contains 20% HK-LP and 80% dextrin, was used in this study. HK-LP was prepared on the basis of the method previously described (5).
Experimental design.
Sixty healthy subjects were enrolled in a randomized, double-blind, placebo-controlled, parallel study. The subjects were randomly assigned by age (middle-aged or elderly) and sex into 2 groups using a sequential series of numbered, sealed envelopes, each containing 1 food ingredient assigned in a computer-randomized fashion. After the assignment, the subjects consumed a hard gelatin capsule containing 50 mg of LP20 (1 capsule/d) or a matching placebo capsule in which dextrin was substituted for LP20 for 12 wk. Throughout the study, the general health of subjects was assessed every 4 wk via direct interviews conducted by the health care provider. Subjects were asked to confirm their compliance with the dietary regimens. Blood samples were collected every 4 wk. Biomarkers for immune function were measured by a clinical laboratory testing company, SRL, every 4 wk. The IgG4:IgG ratio was measured at 0 and 12 wk. Safety assessments such as anthropometric measurements, biochemical examinations of blood, hematological assessments, and urine tests were done at 0 and 12 wk. The study was conducted at PS Clinic (Fukuoka, Japan) from May to August 2005.
Biomarkers for innate immunity.
Peripheral blood mononuclear cells (PBMCs) were isolated by the Ficoll-Conray centrifugation technique (Ficoll-Conray, density = 1.077; centrifuged at 400 × g for 30 min). The NK activity of PBMC was assessed by a standard chromium release method using K562 tumor cells at an effector:target ratio of 20:1 for 3.5 h (12). The neutrophil phagocytosis function was analyzed by flow cytometry (FACSCalibur; Becton Dickinson) after uptake of fluorescein isothiocyanate (FITC)-labeled latex beads (Polyscience) by whole blood. The results are expressed as the percentage of FITC-positive cells, which correspond to cells phagocytosing 1 or more beads, vs. total neutrophils. Cell surface expression of cluster of differentiation (CD)64 on monocytes was analyzed by FACSCalibur after whole blood samples were stained with FITC-conjugated CD64-specific mAb (Becton Dickinson). The results are expressed as the mean fluorescence intensity of CD64-positive monocytes.
Biomarkers for acquired immune functions.
PBMC (5 × 108 cells/L) were cultured with or without suboptimal dose (2 mg/L) of concanavalin A (Con A) for 72 h at 37°C and pulse labeled with 37 kBq of [3H]-thymidine during the last 8 h. DNA synthesis was assessed by measuring thymidine uptake. Percentages of INF-γ and IL-4-producing CD4+ T cells (Th1:Th2 ratio) were determined by single-cell measurement of intracellular cytokines using flow cytometry as described (13). Serum IgG was measured quantitatively by the latex-agglutination method using N-assay TIA IgG-SH (Nittobo) and IgG4 was measured by nephelometry using IgG subclass BS-NIA IgG4 (The Binding Site).
Health-related QOL.
Health-related QOL was assessed every 4 wk using a self-rating questionnaire with 26 items, as shown in Supplemental Table1 (11). Each question was scored from 1 to 3; a higher score indicated a better QOL. Increase in the total QOL score compared with that of the baseline was defined as improved QOL, and unchanged and decreased total QOL scores were defined as unchanged and deteriorated QOL, respectively. QOL changes were scored as follows: improved QOL, 1; unchanged QOL, 0; and deteriorated QOL, −1.
Statistical analysis.
We compared baseline values between groups using the unpaired t test for age and immune functions or the Mann-Whitney U test for total QOL score. We calculated Spearman correlation coefficients between age and immune functions. Because the methods for assessing immune function are based on a highly complicated process including cell culture, we thought that intra-assay variation would increase over time. Therefore, we compared the measurements between groups at the same time point but not within a single individual over time. Percent changes from the baseline in immune functions other than the IgG4:IgG ratio were analyzed by 2-way ANOVA, followed by comparison between groups at each time point using the unpaired t test. We compared QOL change scores between groups by the Mann-Whitney U test. All analyses were performed in all subjects as well as in subjects by age (middle-aged, 40–64 y, and elderly, >64 y) and sex (male and female) using Dr. SPSS software version 8.0J (SPSS Japan). Differences of P < 0.05 were considered significant.
Results
Baseline characteristics.
Before completing the study, 2 subjects in the HK-LP group dropped out, 1 because of chronic ulcerative colitis that had not been recognized as a disease by the subject but was revealed by a medical checkup during the study, and the other as a result of a herniated disk. In addition, 2 subjects in the control group and 1 subject in the HK-LP group who completed the study were excluded from the analysis because the former contracted upper respiratory tract infections and the latter developed herpes labialis, both of which can considerably affect immune function. A total of 55 subjects was included in the statistical analysis. The data of proliferation of 1 subject in the control group was excluded from the analysis, because no response was detected in Con A-stimulated and unstimulated PBMC. Baseline immune characteristics and QOL scores are shown in Table 1. Expression of CD64 on monocytes (P = 0.046) and the unstimulated proliferation of PBMC (P = 0.030) were higher in the HK-LP group than in the control group, and the IgG4:IgG ratio tended to be lower in the HK-LP group (P = 0.077). The other characteristics did not differ between groups. Con A-induced proliferation decreased (r = −0.287, P = 0.028), whereas NK activity increased (r = 0.381, P = 0.003) with age.
Baseline immune characteristics and QOL scores of subjects enrolled in this study1
| . | Control . | HK-LP . | All subjects . |
|---|---|---|---|
| n | 30 | 30 | 60 |
| Male, % | 50 | 50 | 50 |
| Age, y | 55.6 ± 12.8 | 57.0 ± 11.7 | 56.3 ± 12.2 |
| Innate immune function | |||
| NK activity, % cytotoxicity | 35.8 ± 13.9 | 33.2 ± 12.3 | 34.5 ± 13.1 |
| Neutrophil phagocytosis function, %2 | 95.2 ± 1.4 | 95.5 ± 2.3 | 95.4 ± 1.9 |
| CD64 expression on monocytes, Ch3 | 304 ± 57 | 339 ± 77* | 321 ± 69 |
| Acquired immune function | |||
| Unstimulated proliferation, ×103 cpm4 | 0.18 ± 0.06 | 0.23 ± 0.10* | 0.20 ± 0.08 |
| Con A-induced proliferation, ×103 cpm | 13.5 ± 7.9 | 14.1 ± 8.0 | 13.8 ± 7.9 |
| INF-γ-producing cells (Th1 cells), % CD4+ cells | 23.5 ± 10.0 | 21.4 ± 7.7 | 22.5 ± 8.9 |
| IL-4-producing cells (Th2 cells), % CD4+ cells | 2.2 ± 0.9 | 2.6 ± 1.3 | 2.4 ± 1.1 |
| Th1:Th2 ratio | 12.4 ± 6.3 | 10.0 ± 5.4 | 11.2 ± 5.9 |
| IgG4:IgG ratio, ×10−2 | 3.85 ± 3.07 | 2.68 ± 1.82 | 3.26 ± 2.57 |
| Total QOL score | 72.6 ± 5.5 | 71.9 ± 4.4 | 72.3 ± 5.0 |
| . | Control . | HK-LP . | All subjects . |
|---|---|---|---|
| n | 30 | 30 | 60 |
| Male, % | 50 | 50 | 50 |
| Age, y | 55.6 ± 12.8 | 57.0 ± 11.7 | 56.3 ± 12.2 |
| Innate immune function | |||
| NK activity, % cytotoxicity | 35.8 ± 13.9 | 33.2 ± 12.3 | 34.5 ± 13.1 |
| Neutrophil phagocytosis function, %2 | 95.2 ± 1.4 | 95.5 ± 2.3 | 95.4 ± 1.9 |
| CD64 expression on monocytes, Ch3 | 304 ± 57 | 339 ± 77* | 321 ± 69 |
| Acquired immune function | |||
| Unstimulated proliferation, ×103 cpm4 | 0.18 ± 0.06 | 0.23 ± 0.10* | 0.20 ± 0.08 |
| Con A-induced proliferation, ×103 cpm | 13.5 ± 7.9 | 14.1 ± 8.0 | 13.8 ± 7.9 |
| INF-γ-producing cells (Th1 cells), % CD4+ cells | 23.5 ± 10.0 | 21.4 ± 7.7 | 22.5 ± 8.9 |
| IL-4-producing cells (Th2 cells), % CD4+ cells | 2.2 ± 0.9 | 2.6 ± 1.3 | 2.4 ± 1.1 |
| Th1:Th2 ratio | 12.4 ± 6.3 | 10.0 ± 5.4 | 11.2 ± 5.9 |
| IgG4:IgG ratio, ×10−2 | 3.85 ± 3.07 | 2.68 ± 1.82 | 3.26 ± 2.57 |
| Total QOL score | 72.6 ± 5.5 | 71.9 ± 4.4 | 72.3 ± 5.0 |
Values are means ± SD unless otherwise noted. *Different from control, P < 0.05.
FITC-positive cells relative to total neutrophils after the uptake of FITC-labeled latex beads by whole blood.
Channel; fluorescense intensity.
Count per minute.
Baseline immune characteristics and QOL scores of subjects enrolled in this study1
| . | Control . | HK-LP . | All subjects . |
|---|---|---|---|
| n | 30 | 30 | 60 |
| Male, % | 50 | 50 | 50 |
| Age, y | 55.6 ± 12.8 | 57.0 ± 11.7 | 56.3 ± 12.2 |
| Innate immune function | |||
| NK activity, % cytotoxicity | 35.8 ± 13.9 | 33.2 ± 12.3 | 34.5 ± 13.1 |
| Neutrophil phagocytosis function, %2 | 95.2 ± 1.4 | 95.5 ± 2.3 | 95.4 ± 1.9 |
| CD64 expression on monocytes, Ch3 | 304 ± 57 | 339 ± 77* | 321 ± 69 |
| Acquired immune function | |||
| Unstimulated proliferation, ×103 cpm4 | 0.18 ± 0.06 | 0.23 ± 0.10* | 0.20 ± 0.08 |
| Con A-induced proliferation, ×103 cpm | 13.5 ± 7.9 | 14.1 ± 8.0 | 13.8 ± 7.9 |
| INF-γ-producing cells (Th1 cells), % CD4+ cells | 23.5 ± 10.0 | 21.4 ± 7.7 | 22.5 ± 8.9 |
| IL-4-producing cells (Th2 cells), % CD4+ cells | 2.2 ± 0.9 | 2.6 ± 1.3 | 2.4 ± 1.1 |
| Th1:Th2 ratio | 12.4 ± 6.3 | 10.0 ± 5.4 | 11.2 ± 5.9 |
| IgG4:IgG ratio, ×10−2 | 3.85 ± 3.07 | 2.68 ± 1.82 | 3.26 ± 2.57 |
| Total QOL score | 72.6 ± 5.5 | 71.9 ± 4.4 | 72.3 ± 5.0 |
| . | Control . | HK-LP . | All subjects . |
|---|---|---|---|
| n | 30 | 30 | 60 |
| Male, % | 50 | 50 | 50 |
| Age, y | 55.6 ± 12.8 | 57.0 ± 11.7 | 56.3 ± 12.2 |
| Innate immune function | |||
| NK activity, % cytotoxicity | 35.8 ± 13.9 | 33.2 ± 12.3 | 34.5 ± 13.1 |
| Neutrophil phagocytosis function, %2 | 95.2 ± 1.4 | 95.5 ± 2.3 | 95.4 ± 1.9 |
| CD64 expression on monocytes, Ch3 | 304 ± 57 | 339 ± 77* | 321 ± 69 |
| Acquired immune function | |||
| Unstimulated proliferation, ×103 cpm4 | 0.18 ± 0.06 | 0.23 ± 0.10* | 0.20 ± 0.08 |
| Con A-induced proliferation, ×103 cpm | 13.5 ± 7.9 | 14.1 ± 8.0 | 13.8 ± 7.9 |
| INF-γ-producing cells (Th1 cells), % CD4+ cells | 23.5 ± 10.0 | 21.4 ± 7.7 | 22.5 ± 8.9 |
| IL-4-producing cells (Th2 cells), % CD4+ cells | 2.2 ± 0.9 | 2.6 ± 1.3 | 2.4 ± 1.1 |
| Th1:Th2 ratio | 12.4 ± 6.3 | 10.0 ± 5.4 | 11.2 ± 5.9 |
| IgG4:IgG ratio, ×10−2 | 3.85 ± 3.07 | 2.68 ± 1.82 | 3.26 ± 2.57 |
| Total QOL score | 72.6 ± 5.5 | 71.9 ± 4.4 | 72.3 ± 5.0 |
Values are means ± SD unless otherwise noted. *Different from control, P < 0.05.
FITC-positive cells relative to total neutrophils after the uptake of FITC-labeled latex beads by whole blood.
Channel; fluorescense intensity.
Count per minute.
Effects of dietary intervention on innate immunity.
The control and HK-LP groups did not differ in the percent change of NK activity, neutrophil phagocytosis function, or the expression of CD64 on monocytes (Table 2). However, in men (P = 0.028) and in middle-aged subjects (P = 0.054), the percent change in NK activity was greater in the HK-LP group than in the control group (data not shown). On the other hand, in the elderly subjects (P = 0.005) and women (P = 0.054), the percent change in CD64 expression was less in the HK-LP group than in the control group (data not shown).
Percent change from baseline in immune characteristics and QOL change scores in subjects who took either a capsule containing 10 mg of HK-LP or a control capsule each day for 12 wk1
| . | . | . | . | 2-Way ANOVA . | ||
|---|---|---|---|---|---|---|
| . | 4 wk . | 8 wk . | 12 wk . | Intervention . | Time . | Interaction . |
| Innate immune function | % | P-value | ||||
| NK activity | ||||||
| Control | 17.7 ± 28.6 | 17.5 ± 31.9 | 12.8 ± 29.8 | 0.131 | 0.841 | 0.997 |
| HK-LP | 27.7 ± 68.3 | 27.9 ± 45.2 | 24.0 ± 50.5 | |||
| Neutrophil phagocytosis function | ||||||
| Control | −2.4 ± 5.5 | −0.8 ± 2.9 | −1.6 ± 3.7 | 0.614 | 0.416 | 0.314 |
| HK-LP | −1.5 ± 1.9 | −1.7 ± 2.7 | −2.6 ± 3.7 | |||
| CD64 expression on monocytes | ||||||
| Control | 9.7 ± 22.9 | 10.7 ± 31.9 | 9.6 ± 23.8 | 0.164 | 0.968 | 0.956 |
| HK-LP | 4.8 ± 11.8 | 5.6 ± 15.8 | 6.6 ± 14.5 | |||
| Acquired immune function | ||||||
| Unstimulated proliferation | ||||||
| Control | −1 ± 52 | −14 ± 73 | 60 ± 119 | 0.531 | 0.001 | 0.909 |
| HK-LP | −3 ± 42 | −3 ± 54 | 53 ± 142 | |||
| Con A–induced proliferation | ||||||
| Control | −13 ± 53 | −20 ± 60 | 53 ± 88 | 0.036 | <0.001 | 0.894 |
| HK-LP | 9 ± 69 | 18 ± 116 | 84 ± 136 | |||
| INF-γ–producing cells (Th1 cells) | ||||||
| Control | 5.0 ± 18.7 | 5.1 ± 21.6 | 3.0 ± 19.3 | 0.014 | 0.563 | 0.886 |
| HK-LP | 14.5 ± 18.8 | 12.8 ± 21.1 | 8.8 ± 19.3 | |||
| IL-4–producing cells (Th2 cells) | ||||||
| Control | 11.6 ± 44.8 | 31.0 ± 40.3 | 39.1 ± 35.8 | 0.044 | 0.001 | 0.907 |
| HK-LP | −2.1 ± 36.3 | 14.2 ± 52.9 | 29.5 ± 42.2 | |||
| Th1:Th2 ratio | ||||||
| Control | 8.2 ± 48.5 | −10.8 ± 43.3 | −21.4 ± 25.4 | 0.002 | <0.001 | 0.721 |
| HK-LP | 33.4 ± 58.1 | 14.8 ± 46.0* | −7.6 ± 32.7 | |||
| IgG4:IgG ratio | ||||||
| Control | — | — | 4.2 ± 21.9 | — | — | — |
| HK-LP | — | — | −0.8 ± 17.6 | |||
| QOL change scores2 | ||||||
| Control | 0.11 ± 0.88 | −0.04 ± 0.92 | −0.07 ± 0.90 | — | — | — |
| HK-LP | 0.11 ± 0.89 | 0.44 ± 0.75* | 0.33 ± 0.83 | |||
| . | . | . | . | 2-Way ANOVA . | ||
|---|---|---|---|---|---|---|
| . | 4 wk . | 8 wk . | 12 wk . | Intervention . | Time . | Interaction . |
| Innate immune function | % | P-value | ||||
| NK activity | ||||||
| Control | 17.7 ± 28.6 | 17.5 ± 31.9 | 12.8 ± 29.8 | 0.131 | 0.841 | 0.997 |
| HK-LP | 27.7 ± 68.3 | 27.9 ± 45.2 | 24.0 ± 50.5 | |||
| Neutrophil phagocytosis function | ||||||
| Control | −2.4 ± 5.5 | −0.8 ± 2.9 | −1.6 ± 3.7 | 0.614 | 0.416 | 0.314 |
| HK-LP | −1.5 ± 1.9 | −1.7 ± 2.7 | −2.6 ± 3.7 | |||
| CD64 expression on monocytes | ||||||
| Control | 9.7 ± 22.9 | 10.7 ± 31.9 | 9.6 ± 23.8 | 0.164 | 0.968 | 0.956 |
| HK-LP | 4.8 ± 11.8 | 5.6 ± 15.8 | 6.6 ± 14.5 | |||
| Acquired immune function | ||||||
| Unstimulated proliferation | ||||||
| Control | −1 ± 52 | −14 ± 73 | 60 ± 119 | 0.531 | 0.001 | 0.909 |
| HK-LP | −3 ± 42 | −3 ± 54 | 53 ± 142 | |||
| Con A–induced proliferation | ||||||
| Control | −13 ± 53 | −20 ± 60 | 53 ± 88 | 0.036 | <0.001 | 0.894 |
| HK-LP | 9 ± 69 | 18 ± 116 | 84 ± 136 | |||
| INF-γ–producing cells (Th1 cells) | ||||||
| Control | 5.0 ± 18.7 | 5.1 ± 21.6 | 3.0 ± 19.3 | 0.014 | 0.563 | 0.886 |
| HK-LP | 14.5 ± 18.8 | 12.8 ± 21.1 | 8.8 ± 19.3 | |||
| IL-4–producing cells (Th2 cells) | ||||||
| Control | 11.6 ± 44.8 | 31.0 ± 40.3 | 39.1 ± 35.8 | 0.044 | 0.001 | 0.907 |
| HK-LP | −2.1 ± 36.3 | 14.2 ± 52.9 | 29.5 ± 42.2 | |||
| Th1:Th2 ratio | ||||||
| Control | 8.2 ± 48.5 | −10.8 ± 43.3 | −21.4 ± 25.4 | 0.002 | <0.001 | 0.721 |
| HK-LP | 33.4 ± 58.1 | 14.8 ± 46.0* | −7.6 ± 32.7 | |||
| IgG4:IgG ratio | ||||||
| Control | — | — | 4.2 ± 21.9 | — | — | — |
| HK-LP | — | — | −0.8 ± 17.6 | |||
| QOL change scores2 | ||||||
| Control | 0.11 ± 0.88 | −0.04 ± 0.92 | −0.07 ± 0.90 | — | — | — |
| HK-LP | 0.11 ± 0.89 | 0.44 ± 0.75* | 0.33 ± 0.83 | |||
Values are means ± SD, n = 27. HK-LP group, n = 27. *Different from the control group at that time, P < 0.05.
The QOL change scores at each time point relative to baseline were graded as follows: 1, improved; 0, unchanged; −1, deteriorated.
Percent change from baseline in immune characteristics and QOL change scores in subjects who took either a capsule containing 10 mg of HK-LP or a control capsule each day for 12 wk1
| . | . | . | . | 2-Way ANOVA . | ||
|---|---|---|---|---|---|---|
| . | 4 wk . | 8 wk . | 12 wk . | Intervention . | Time . | Interaction . |
| Innate immune function | % | P-value | ||||
| NK activity | ||||||
| Control | 17.7 ± 28.6 | 17.5 ± 31.9 | 12.8 ± 29.8 | 0.131 | 0.841 | 0.997 |
| HK-LP | 27.7 ± 68.3 | 27.9 ± 45.2 | 24.0 ± 50.5 | |||
| Neutrophil phagocytosis function | ||||||
| Control | −2.4 ± 5.5 | −0.8 ± 2.9 | −1.6 ± 3.7 | 0.614 | 0.416 | 0.314 |
| HK-LP | −1.5 ± 1.9 | −1.7 ± 2.7 | −2.6 ± 3.7 | |||
| CD64 expression on monocytes | ||||||
| Control | 9.7 ± 22.9 | 10.7 ± 31.9 | 9.6 ± 23.8 | 0.164 | 0.968 | 0.956 |
| HK-LP | 4.8 ± 11.8 | 5.6 ± 15.8 | 6.6 ± 14.5 | |||
| Acquired immune function | ||||||
| Unstimulated proliferation | ||||||
| Control | −1 ± 52 | −14 ± 73 | 60 ± 119 | 0.531 | 0.001 | 0.909 |
| HK-LP | −3 ± 42 | −3 ± 54 | 53 ± 142 | |||
| Con A–induced proliferation | ||||||
| Control | −13 ± 53 | −20 ± 60 | 53 ± 88 | 0.036 | <0.001 | 0.894 |
| HK-LP | 9 ± 69 | 18 ± 116 | 84 ± 136 | |||
| INF-γ–producing cells (Th1 cells) | ||||||
| Control | 5.0 ± 18.7 | 5.1 ± 21.6 | 3.0 ± 19.3 | 0.014 | 0.563 | 0.886 |
| HK-LP | 14.5 ± 18.8 | 12.8 ± 21.1 | 8.8 ± 19.3 | |||
| IL-4–producing cells (Th2 cells) | ||||||
| Control | 11.6 ± 44.8 | 31.0 ± 40.3 | 39.1 ± 35.8 | 0.044 | 0.001 | 0.907 |
| HK-LP | −2.1 ± 36.3 | 14.2 ± 52.9 | 29.5 ± 42.2 | |||
| Th1:Th2 ratio | ||||||
| Control | 8.2 ± 48.5 | −10.8 ± 43.3 | −21.4 ± 25.4 | 0.002 | <0.001 | 0.721 |
| HK-LP | 33.4 ± 58.1 | 14.8 ± 46.0* | −7.6 ± 32.7 | |||
| IgG4:IgG ratio | ||||||
| Control | — | — | 4.2 ± 21.9 | — | — | — |
| HK-LP | — | — | −0.8 ± 17.6 | |||
| QOL change scores2 | ||||||
| Control | 0.11 ± 0.88 | −0.04 ± 0.92 | −0.07 ± 0.90 | — | — | — |
| HK-LP | 0.11 ± 0.89 | 0.44 ± 0.75* | 0.33 ± 0.83 | |||
| . | . | . | . | 2-Way ANOVA . | ||
|---|---|---|---|---|---|---|
| . | 4 wk . | 8 wk . | 12 wk . | Intervention . | Time . | Interaction . |
| Innate immune function | % | P-value | ||||
| NK activity | ||||||
| Control | 17.7 ± 28.6 | 17.5 ± 31.9 | 12.8 ± 29.8 | 0.131 | 0.841 | 0.997 |
| HK-LP | 27.7 ± 68.3 | 27.9 ± 45.2 | 24.0 ± 50.5 | |||
| Neutrophil phagocytosis function | ||||||
| Control | −2.4 ± 5.5 | −0.8 ± 2.9 | −1.6 ± 3.7 | 0.614 | 0.416 | 0.314 |
| HK-LP | −1.5 ± 1.9 | −1.7 ± 2.7 | −2.6 ± 3.7 | |||
| CD64 expression on monocytes | ||||||
| Control | 9.7 ± 22.9 | 10.7 ± 31.9 | 9.6 ± 23.8 | 0.164 | 0.968 | 0.956 |
| HK-LP | 4.8 ± 11.8 | 5.6 ± 15.8 | 6.6 ± 14.5 | |||
| Acquired immune function | ||||||
| Unstimulated proliferation | ||||||
| Control | −1 ± 52 | −14 ± 73 | 60 ± 119 | 0.531 | 0.001 | 0.909 |
| HK-LP | −3 ± 42 | −3 ± 54 | 53 ± 142 | |||
| Con A–induced proliferation | ||||||
| Control | −13 ± 53 | −20 ± 60 | 53 ± 88 | 0.036 | <0.001 | 0.894 |
| HK-LP | 9 ± 69 | 18 ± 116 | 84 ± 136 | |||
| INF-γ–producing cells (Th1 cells) | ||||||
| Control | 5.0 ± 18.7 | 5.1 ± 21.6 | 3.0 ± 19.3 | 0.014 | 0.563 | 0.886 |
| HK-LP | 14.5 ± 18.8 | 12.8 ± 21.1 | 8.8 ± 19.3 | |||
| IL-4–producing cells (Th2 cells) | ||||||
| Control | 11.6 ± 44.8 | 31.0 ± 40.3 | 39.1 ± 35.8 | 0.044 | 0.001 | 0.907 |
| HK-LP | −2.1 ± 36.3 | 14.2 ± 52.9 | 29.5 ± 42.2 | |||
| Th1:Th2 ratio | ||||||
| Control | 8.2 ± 48.5 | −10.8 ± 43.3 | −21.4 ± 25.4 | 0.002 | <0.001 | 0.721 |
| HK-LP | 33.4 ± 58.1 | 14.8 ± 46.0* | −7.6 ± 32.7 | |||
| IgG4:IgG ratio | ||||||
| Control | — | — | 4.2 ± 21.9 | — | — | — |
| HK-LP | — | — | −0.8 ± 17.6 | |||
| QOL change scores2 | ||||||
| Control | 0.11 ± 0.88 | −0.04 ± 0.92 | −0.07 ± 0.90 | — | — | — |
| HK-LP | 0.11 ± 0.89 | 0.44 ± 0.75* | 0.33 ± 0.83 | |||
Values are means ± SD, n = 27. HK-LP group, n = 27. *Different from the control group at that time, P < 0.05.
The QOL change scores at each time point relative to baseline were graded as follows: 1, improved; 0, unchanged; −1, deteriorated.
Effects of dietary intervention on acquired immune function.
The percent change in Con A-induced proliferation was greater in the HK-LP group than in the control group (P = 0.036), whereas the percent change of unstimulated proliferation did not differ between groups (Table 2). A similar effect of HK-LP on Con A-stimulated proliferation was also observed in middle-aged subjects (P = 0.077, data not shown). The percent change in the Th1:Th2 ratio was higher in the HK-LP group than in the control group (P = 0.002) because of a higher percent change in Th1 cells and lower percent change in Th2 cells in the HK-LP group than in the control group (Table 2). A similar effect of HK-LP on the Th1:Th2 ratio was also observed in middle-aged subjects (P = 0.001) and men (P < 0.001; data not shown). The percent change in IgG4:IgG ratio did not differ between groups (Table 2). However, in women, the percent change was less in the HK-LP group than in the control group (P = 0.070, data not shown). The time of sampling affected both Con A-induced proliferation (P < 0.001) and the Th1:Th2 ratio (P = 0.001) (Table 2).
Effects of dietary intervention on health-related QOL.
The QOL change score, the degree of improvement in QOL, was higher at 8 wk (P = 0.049) and tended to be higher at 12 wk (P = 0.092) in the HK-LP group than in the control group (Table 2).
Safety assessments.
Three adverse events in the control group and 6 in the HK-LP group were recorded during the study. The adverse events other than mentioned above were knee arthralgia in the control group and Meniere's syndrome, dizziness, and acute urticaria in the HK-LP group. All adverse events were judged to be unrelated to the dietary intervention. Among the safety assessments that we performed, the percent change in lymphocyte count was greater in the HK-LP group than in the control groups (P < 0.05), but the changes were within the range of reference values.
Discussion
In this study, the healthy subjects who took HK-LP daily sustained increases in Con A-induced proliferation of PBMC and the Th1:Th2 ratio of CD4+ T cells from 4 wk after the start of dietary intervention. Subsequently, the health-related QOL also improved from wk 8 in the HK-LP supplemented group. HK-LP has been shown to be a potent inducer of IL-12 in vitro as well as in vivo in mice and is therefore effective for the prophylaxis and treatment of the disease evoked by the weaker Th1 type immune responses. Our results in this study illustrate the possibility that the effects of HK-LP demonstrated in animal experiments may also occur in humans.
Because there are many reports of improvement of immune function by food-derived components in healthy individuals, systematic analysis of these reports enables us to advocate reliable biomarkers for assessing the immunomodulating effects of food-derived substances. By doing this, Kaminogawa et al. (9) have advocated general biomarkers for assessing immune function. To examine the effects of HK-LP intake on immune function in healthy individuals, we measured such general biomarkers, including phagocytic activity, NK activity and the proliferative response of T cells. Among these biomarkers, the proliferative response of T cells was considerably augmented by HK-LP. The acquired immunity such as the proliferative response of T cells reportedly decreases as a result of aging and of stress (14–18). In accordance with these observations, Con A-induced proliferation was decreased in association with aging in this study. The poorer acquired immunity was reported to correlate with the risk of various infectious diseases (14–18). Therefore, the fact that intake of HK-LP augmented the proliferative response of T cells during the dietary period suggests that HK-LP is beneficial for the prevention of infectious diseases.
Several lines of evidence show that dietary consumption of probiotic lactic acid bacteria can enhance the activity of NK cells in healthy adult subjects (19,20). Although the intake of HK-LP did not affect NK activity in all subjects (Table 2), it did affect NK activity in men and the middle-aged subjects. We do not know why other subjects did not respond to HK-LP, but the reason may be related to the strong influence of the menstrual cycle on NK activity in females (21) and to age-related changes in the responsiveness to external stimuli in the elderly. HK-LP did not enhance the neutrophil phagocytosis function, although lactic acid bacteria have been reported to enhance phagocytic activity (19,20). We assessed the neutrophil phagocytosis function by a conventional clinical method for assessing neutrophil dysfunction, in which functional neutrophils that could phagocytose 1 or more beads exceeded 90% of the total neutrophils in most healthy subjects. Thus, the method is not appropriate for evaluating the effect of immunomodulators on phagocytic activity in healthy subjects. To clarify the effects of HK-LP on innate immunity in healthy subjects, further examinations are needed.
We examined the effects of the intake of HK-LP on Th1-related immune function by determining the Th1:Th2 ratio of CD4+ T cells. Intake of HK-LP considerably augmented the Th1:Th2 ratio in healthy subjects, as was observed previously in a study in which a number of lactic acid bacteria skewed T cells toward Th1 polarization in vitro (7). This result suggests that HK-LP may decrease risk for the development of infectious and allergic disease associated with a low Th1:Th2 ratio. We also measured the expression of CD64 on monocytes and the serum IgG4:IgG ratio as a candidate for a biomarker for Th1-related immune function on the basis of observation that in vivo treatment with INF-γ in healthy human subjects led to an increased expression of CD64 (FcγRI) on monocytes (22) and that higher Th1:Th2 ratios enhanced IgG4 antibody production from PBMC in subjects who responded to bee venom (23). However, intake of HK-LP did not affect either the expression of CD64 on monocytes or the serum IgG4:IgG ratio in all subjects (Table 2). Therefore, it seems that these biomarkers are not appropriate for assessing Th1-related immune function in healthy subjects.
In the measurements of either Con A-induced proliferation or the Th1:Th2 ratio, a time point effect was observed (Table 2). Because the methods for measurement of these biomarkers are based on a highly complicated process including cell culture, measurement noise must be considered. Intra-assay variation is thought to be lower in measurements at the same time point than in those at different time points. In addition, measurement reliability can be enhanced by measuring a control specimen. Therefore, we considered that the comparison between groups at the same time point was reliable but that those over time were not. However, we could not exclude the possibility that Con A-induced proliferation and Th1:Th2 ratio varied seasonally.
QOL has been defined by the WHO as “the state of complete physical, mental, and social well-being and not merely the absence of disease or infirmity.” Schipper suggested that QOL has 4 components: physical and occupational functions, social interaction, psychological state, and somatic sensation (24). To investigate the effects of HK-LP on health, we measured health-related QOL using a self-rating questionnaire aimed at both physical and psychological states. The degrees of improvement in the health-related QOL were higher from wk 8 in the HK-LP group following the augmentation of the immune function such as Con A-induced proliferation and the Th1:Th2 ratio, which were seen from wk 4. These results suggest that improvement of the health-related QOL by HK-LP is accomplished by the enhancement of immune function in healthy subjects.
In conclusion, we demonstrated that a daily intake of HK-LP enhanced acquired immunity, especially Th1-related immune functions in healthy subjects, in addition to inducing subsequent improvements of the health-related QOL. Our results suggest that intake of HK-LP may be useful for prevention and treatment of infectious and allergic diseases induced by a weak Th1-type immune response.
Literature Cited
Abbreviations
- CD
cluster of differentiation
- Con A
concanavalin A
- FITC
fluorescein isothiocyanate
- HK-LP
heat-killed Lactobacillus plantarum L-137
- NK
natural killer
- PBMC
peripheral blood mononuclear cell
- QOL
quality of life
- Th
T helper
- diet therapy
- immune response
- adult
- allergens
- biological markers
- concanavalin a
- food
- heat (physical force)
- immunity, natural
- interleukin-12
- interleukin-4
- lactobacillus
- monocytes
- neutrophils
- phagocytosis
- t-lymphocytes
- immunoglobulin g
- immunoglobulin e
- mice
- quality of life
- immunologic function
- tumor growth
- acquired immunity
- parallel study
- immunoglobulin g4
- peripheral blood mononuclear cell
- clusters of differentiation
- cd64 antigen
