Efficacy of Lactococcus lactis strain plasma (LC-Plasma) in easing symptoms in patients with mild COVID-19: protocol for an exploratory, multicentre, double-blinded, randomised controlled trial (PLATEAU study)

Introduction The COVID-19 pandemic has been a major concern worldwide; however, easily accessible treatment options for patients with mild COVID-19 remain limited. Since the oral intake of Lactococcus lactis strain plasma (LC-Plasma) enhances both the innate and acquired immune systems through the activation of plasmacytoid dendritic cells (pDCs), we hypothesised that the oral intake of LC-Plasma could aid the relief or prevention of symptoms in patients with asymptomatic or mild COVID-19. Methods and analysis This is an exploratory, multicentre, double-blinded, randomised, placebo-controlled trial. This study was initiated in December 2021 and concludes in April 2023. The planned number of enrolled subjects is 100 (50 subjects×2 groups); subject enrolment will be conducted until October 2022. Patients with asymptomatic or mild COVID-19 will be enrolled and randomly assigned in a 1:1 ratio to group A (oral intake of LC-Plasma-containing capsule, 200 mg/day, for 14 days) or group B (oral intake of placebo capsule, for 14 days). The primary endpoint is the change in subjective symptoms measured by the severity score. Secondary endpoints include SARS-CoV-2 viral loads, biomarkers for pDC activation, serum SARS-CoV-2-specific antibodies, serum cytokines, interferon and interferon-inducible antiviral effectors and the proportion of subjects with emergency room visits to medical institutions or who are hospitalised. Ethics and dissemination The study protocol was approved by the Clinical Research Review Board of Nagasaki University, in accordance with the Clinical Trials Act of Japan. The study will be conducted in accordance with the Declaration of Helsinki, the Clinical Trials Act, and other current legal regulations in Japan. Written informed consent will be obtained from all the participants. The results of this study will be reported in journal publications

Spherical Lactic Acid Bacteria Activate Plasmacytoid Dendritic Cells Immunomodulatory Function via TLR9-Dependent Crosstalk with Myeloid Dendritic Cells

Plasmacytoid dendritic cells (pDC) are a specialized sensor of viral and bacterial nucleic acids and a major producer of IFN-α that promotes host defense by priming both innate and acquired immune responses. Although synthetic Toll-like receptor (TLR) ligands, pathogenic bacteria and viruses activate pDC, there is limited investigation of non-pathogenic microbiota that are in wide industrial dietary use, such as lactic acid bacteria (LAB). In this study, we screened for LAB strains, which induce pDC activation and IFN-α production using murine bone marrow (BM)-derived Flt-3L induced dendritic cell culture. Microbial strains with such activity on pDC were absent in a diversity of bacillary strains, but were observed in certain spherical species (Lactococcus, Leuconostoc, Streptococcus and Pediococcus), which was correlated with their capacity for uptake by pDC. Detailed study of Lactococcus lactis subsp. lactis JCM5805 and JCM20101 revealed that the major type I and type III interferons were induced (IFN-α, -β, and λ). IFN-α induction was TLR9 and MyD88-dependent; a slight impairment was also observed in TLR4-/- cells. While these responses occurred with purified pDC, IFN-α production was synergistic upon co-culture with myeloid dendritic cells (mDC), an interaction that required direct mDC-pDC contact. L. lactis strains also stimulated expression of immunoregulatory receptors on pDC (ICOS-L and PD-L1), and accordingly augmented pDC induction of CD4+CD25+FoxP3+ Treg compared to the Lactobacillus strain. Oral administration of L. lactis JCM5805 induced significant activation of pDC resident in the intestinal draining mesenteric lymph nodes, but not in a remote lymphoid site (spleen). Taken together, certain non-pathogenic spherical LAB in wide dietary use has potent and diverse immunomodulatory effects on pDC potentially relevant to anti-viral immunity and chronic inflammatory disease

Oral administration of Lactococcus lactis subsp. lactis JCM5805 enhances lung immune response resulting in protection from murine parainfluenza virus infection.

When activated by viral infection, plasmacytoid dendritic cells (pDCs) play a primary role in the immune response through secretion of IFN-α. Lactococcus lactis subsp. lactis JCM5805 (JCM5805) is a strain of lactic acid bacteria (LAB) that activates murine and human pDCs to express type I and type III interferons (IFNs). JCM5805 has also been shown to activate pDCs via a Toll-like receptor 9 (TLR9) dependent pathway. In this study, we investigated the anti-viral effects of oral administration of JCM5805 using a mouse model of murine parainfluenza virus (mPIV1) infection. JCM5805-fed mice showed a drastic improvement in survival rate, prevention of weight loss, and reduction in lung histopathology scores compared to control mice. We further examined the mechanism of anti-viral effects elicited by JCM5805 administration using naive mice. Microscopic observations showed that JCM5805 was incorporated into CD11c+ immune cells in Peyer's patches (PP) and PP pDCs were significantly activated and the expression levels of IFNs were significantly increased. Interestingly, nevertheless resident pDCs at lung were not activated and expressions levels of IFNs at whole lung tissue were not influenced, the expressions of anti-viral factors induced by IFNs, such as Isg15, Oasl2, and Viperin, at lung were up-regulated in JCM5805-fed mice compared to control mice. Therefore expressed IFNs from intestine might be delivered to lung and IFN stimulated genes might be induced. Furthermore, elevated expressions of type I IFNs from lung lymphocytes were observed in response to mPIV1 ex vivo stimulation in JCM5805-fed mice compared to control. This might be due to increased ratio of pDCs located in lung were significantly increased in JCM5805 group. Taken together, a specific LAB strain might be able to affect anti-viral immunological profile in lung via activation of intestinal pDC leading to enhanced anti-viral phenotype in vivo

Effect of Heat-Killed Lactobacillus paracasei KW3110 Ingestion on Ocular Disorders Caused by Visual Display Terminal (VDT) Loads: A Randomized, Double-Blind, Placebo-Controlled Parallel-Group Study

Background: Visual display terminals (VDTs) emitting blue light can cause ocular disorders including eye fatigue. Some dietary constituents have been reported to be effective in improving ocular disorders while few clinical studies have been performed. We evaluated the effects of heat-killed Lactobacillus paracasei KW 3110 on improving ocular disorders and symptoms of eye fatigue among healthy human subjects with VDT loads. Methods: In vitro, the effect of L. paracasei KW3110 on blue light-induced human retinal pigment epithelial (ARPE-19) cell damage. For clinical studies, 62 healthy Japanese volunteers of 35 to 45 years of age who had experienced eye fatigue were randomized into two groups and given a placebo or L. paracasei KW3110-containing supplements for eight weeks. The primary endpoint was changes in VDT load-induced eye fatigue as determined by critical flicker frequency four and eight weeks after the start of supplementation. Results: In vitro, blue light-induced human retinal cell death was suppressed with the culture supernatants of cells treated with L. paracasei KW3110. In clinical study, the VDT load-induced reduction of critical flicker frequency tended to be milder in the L. paracasei KW3110 group when compared with the placebo group during the fourth week. Subgroup analysis classified by the degree of eye fatigue showed that the VDT load-induced reduction of critical flicker frequency was significantly better in the high-level eye fatigue subjects from the L. paracasei KW3110 group when compared with the placebo group during the fourth week (p = 0.020). Conclusions: L. paracasei KW3110 suppressed blue light-induced retinal pigment epithelial cell death. In the clinical study, ingestion of L. paracasei KW3110 had a potential to improve eye fatigue induced by VDT loads especially high levels of eye fatigue. However, further studies should be required to show more dependable clinical efficacy of L. paracasei KW3110

<i>Lactobacillus paracasei</i> KW3110 Prevents Blue Light-Induced Inflammation and Degeneration in the Retina

Age-related macular degeneration and retinitis pigmentosa are leading causes of blindness and share a pathological feature, which is photoreceptor degeneration. To date, the lack of a potential treatment to prevent such diseases has raised great concern. Photoreceptor degeneration can be accelerated by excessive light exposure via an inflammatory response; therefore, anti-inflammatory agents would be candidates to prevent the progress of photoreceptor degeneration. We previously reported that a lactic acid bacterium, Lactobacillus paracasei KW3110 (L. paracasei KW3110), activated macrophages suppressing inflammation in mice and humans. Recently, we also showed that intake of L. paracasei KW3110 could mitigate visual display terminal (VDT) load-induced ocular disorders in humans. However, the biological mechanism of L. paracasei KW3110 to retain visual function remains unclear. In this study, we found that L. paracasei KW3110 activated M2 macrophages inducing anti-inflammatory cytokine interleukin-10 (IL-10) production in vitro using bone marrow-derived M2 macrophages. We also show that IL-10 gene expression was significantly increased in the intestinal immune tissues 6 h after oral administration of L. paracasei KW3110 in vivo. Furthermore, we demonstrated that intake of L. paracasei KW3110 suppressed inflammation and photoreceptor degeneration in a murine model of light-induced retinopathy. These results suggest that L. paracasei KW3110 may have a preventive effect against degrative retinal diseases

Activation of pDCs in intestine by JCM5805 administration.

<p>Healthy C57BL / 6J mice were divided into control and JCM5805 groups (n = 4 in each group), and mice in the JCM5805 group were orally administered JCM5808 daily for 2 weeks. A. Low density cells prepared from PP of each group were analyzed by FACS. Expression level of cell surface activation marker was evaluated for MHC class II as median fluorescence intensities (M.F.I.) in left panel. Ratio of pDCs to total population was shown in right panel. pDCs was defined as “CD3⁻ Siglec-H⁺ CD11c⁺ in total population”. Short line represents the mean values. *<i>P</i><0.05 (Student’s t test). B, Total mRNA was extracted from PP pDCs from mice in the control (open columns) and JCM5805 groups (dot columns) (n = 8 in each group). <i>Ifnα</i> and <i>Ifnβ</i> gene expressions were measured by qRT-PCR and normalized to <i>Gapdh</i> gene expression. Data are shown as mean ± SD. *<i>P</i><0.05 (Student’s t test). These data are representative of three independent experiments. Each data are mean ± SD.</p

Lung histopathology of mPIV1-infected mice.

<p>A. Representative hematoxylin and eosin (H & E)-stained sections of lung tissues from control and JCM5805 group mice (6 mice per group). Lung tissues were prepared from mice 3 days after infection. Scale bars, 300 μm. B. Histological scoring of lung tissues from mPIV1-infected mice belong to control (open columns) and JCM5805 (dot columns) group. Sections were scored at four levels as follows: 0, no symptoms; 1, low pathogenicity; 2, medium pathogenicity; 3, high pathogenicity. The mean ± SD of the tissues in each group is shown. *<i>P</i><0.05, **<i>P</i><0.01 (Mann-Whitney U test). The data shown is representative of two independent experiments.</p

Effects of JCM5805 on mPIV1 infection.

<p>A. Experimental procedure of mPIV1 infection. Mice in the control and JCM5805 groups were fed diet with or without 1 mg / mouse / day of JCM5808 during the study period (day -14 to 15). Mice were intranasally infected with mPIV1 on day 0. On 3 days post-mPIV1 infection, six mice were sacrificed from each group for lung histopathology. Thereafter survival rate, body weight and clinical scores were investigated with remained control mice n = 12, and JCM5805 mice n = 13. B. Survival rate of mice infected with mPIV1. The control (circle) and JCM5805 (square) groups consisted of 12 and 13 mice, respectively. The survival of each animal was monitored daily. <i>P</i><0.001 (Log-Rank test). C. Body weight of mice infected with mPIV1. The control (circle) and JCM5805 (square) groups consisted of 12 and 13 mice, respectively. The body weight of each surviving animal was measured daily. The body weight values are shown as mean ± SD. *<i>P</i><0.05, **<i>P</i><0.01 (Student’s t test). The data shown is representative of two independent experiments.</p