COVID-19 infection and ginseng: Predictive influenza virus strains and non-predictive COVID-19 vaccine strains.

Vaccines help protect people from infections. However, Coronavirus 2019 (COVID-19) vaccinees often still become infected with COVID-19 variants (breakthrough infections) and may go on to suffer from long COVID symptoms due to short-lasting immunity and less-effective protection provided by available vaccines. Moreover, the current COVID-19 vaccines do not prevent viral transmission and ward off only about 15% of breakthrough infections. To prepare more effective vaccines, it is essential to predict the viral strains that will be circulating based on available epidemiological data. The World Health Organization recommends in advance which influenza strains are expected to be prevalent during influenza season to guide the production of influenza vaccines by pharmaceutical companies. However, future emerging COVID-19 strain(s) have not been possible to predict since no sound epidemiological information has been established. Thus, for more effective protection, immune stimulators alone or in combination with vaccines would be preferable to protect people from COVID-19 infection. One of those remedies would be ginseng, which has been used for potentiating immunity in the past.

Immunization with a Pneumococcal pep27 Mutant Strain Alleviates Atopic Dermatitis through the Upregulation of Regulatory T-Cell Activity and Epithelial Barrier Function and Suppressing TSLP Expression.

Atopic dermatitis (AD) is an inflammatory disease driven in part by type 2 helper T (Th2) cytokines and skin barrier disruption alleviating the entry of allergens. Thymic stromal lymphopoietin (TSLP), an epithelial cell‒derived cytokine, is known to aggravate AD symptoms by activating Th2. In addition, regulatory T cells (Tregs) inhibit inflammatory cells such as Th2. However, the relationship between TSLP and Tregs in AD is unclear. A murine dermatitis model was induced by applying oxazolone to the ear skin of mice. Prophylactic and therapeutic responses were analyzed by immunizing mice intranasally with a pneumococcal pep27 mutant (Δpep27 mutant), attenuated strain by reducing the virulence of a pathogen. Intranasal immunization with a pneumococcal pep27 mutant could elicit anti-inflammatory Treg-relevant factors and epithelial barrier genes (loricrin, involucrin, filaggrin, and small proline-rich repeat proteins). Thus, pneumococcal pep27-mutant immunization suppressed epidermal collapse, IgE, TSLP, and upregulation of Th2 expression by upregulating Treg activity. In contrast, Treg inhibition aggravated AD symptoms through the upregulation of TSLP and Th2 and the repression of epithelial barrier function compared with that of the noninhibited pneumococcal Δpep27-mutant group. Taken together, immunization with pneumococcal Δpep27 mutant upregulated Treg and epithelial barrier function and inhibited TSLP and Th2 to relieve AD symptoms.

Pep27 Mutant Immunization Inhibits Caspase-14 Expression to Alleviate Inflammatory Bowel Disease via Treg Upregulation.

Inflammatory bowel disease (IBD) is a highly prevalent gut inflammatory disorder. Complicated clinical outcomes prolong the use of conventional therapy and often lead to compromised immunity followed by adverse events and high relapse rates. Thus, a profound medical intervention is required. Previously, intranasal immunization of pneumococcal pep27 mutant (Δpep27) exhibited long-lasting protection against immune-related disorders. System biology analysis has predicted an inverse correlation between Δpep27 immunization and gastroenteritis. Recently, we established that Δpep27-elicited Tregs repressed Wnt5a expression and enhanced barrier integrity, suggesting the restoration of immunological tolerance. Therefore, we evaluated whether Δpep27 can alleviate IBD. Δpep27 dose-dependent response was analyzed in dextran sulfate sodium-induced mice using transcriptome analysis. Pro- and anti-inflammatory signatures were cross-correlated by quantitative PCR and western blot analyses. To address the hierarchy regulating the activity of caspase-14, an undefined marker in IBD, and regulatory T cells (Tregs), antibody-based neutralization studies were conducted. Fecal microbiome profiles were analyzed by 16S rRNA pyrosequencing. Δpep27 significantly attenuated dextran sulfate sodium-induced oxidative stress parameters, proinflammatory cytokines, caspase-14 expression level, and upregulated tight junction, anti-inflammatory genes IL-10 and TGF-ß1 via upregulation of Tregs to restore healthy gut microbiota. Neutralization studies unveiled that ∆pep27 had a remedial effect via Treg upregulation. Caspase-14, being an important mediator in the pathogenesis of IBD, can be an alternate therapeutic target in IBD. ∆pep27-increased Tregs repressed caspase-14 expression and reversed gut microbial dysbiosis, aiding to re-establish immunological tolerance.

Pneumococcal pep27-mutant inhibits Wnt5a expression via the regulation of T helper cells to attenuate colitis.

Inflammatory bowel disease (IBD) is a chronic gut inflammatory disease characterized by extensive colitis and remission of the symptoms. The incidence rate and prevalence of IBD are increasing worldwide; IBD affects millions of people, has poorly defined etiology, and often results in a failure of pharmacological interventions. Regardless of the cause, mucosal healing is indispensable for the regeneration of inflamed mucosa to ensure intestinal homeostasis. Intranasal immunization with the pneumococcal pep27 mutant (Δpep27) has been reported as an avirulent and live vaccine that has been proposed to suppress immune-regulated disorders, eliciting long-lasting immunity. The dose-dependent activity of Δpep27 in the lungs was measured by transcriptome analysis to investigate the long-lasting immunogenic response against IBD. Novel therapeutic targets based on the modulation of Wnt signaling and T regulatory cells interconnected with other signaling cascades in the context of IBD were investigated by qPCR and immunoblotting. M1/M2 macrophages were quantified by FACS analysis. Dextran sulfate sodium-induced colitis induced significant upregulation of Th2 and Th17 as well as noncanonical Wnt5, which subsequently inhibited regulatory T (Treg) expression. In contrast, Δpep27 immunization significantly attenuated the levels of Wnt5, proinflammatory cytokines, oxidative stress parameters, and infiltration of inflammatory cells and enhanced barrier integrity via T helper cell homeostasis and upregulation of M2 macrophages. The data of the present study suggested that Δpep27-elicited Tregs were able to repress Wnt5a expression, assisting with the restoration of immunological tolerance and providing a robust regenerative and antioxidant milieu.

Pneumococcal Δpep27 Immunization Attenuates TLRs and NLRP3 Expression and Relieves Murine Ovalbumin-Induced Allergic Rhinitis.

Allergic rhinitis (AR), one of the most common inflammatory diseases, is caused by immunoglobulin E (IgE)-mediated reactions against inhaled allergens. AR involves mucosal inflammation driven by type 2 helper T (Th2) cells. Previously, it was shown that the Streptococcus pneumoniae pep27 mutant (Δpep27) could prevent and treat allergic asthma by reducing Th2 responses. However, the underlying mechanism of Δpep27 immunization in AR remains undetermined. Here, we investigated the role of Δpep27 immunization in the development and progression of AR and elucidated potential mechanisms. In an ovalbumin (OVA)-induced AR mice model, Δpep27 alleviated allergic symptoms (frequency of sneezing and rubbing) and reduced TLR2 and TLR4 expression, Th2 cytokines, and eosinophil infiltration in the nasal mucosa. Mechanistically, Δpep27 reduced the activation of the NLRP3 inflammasome in the nasal mucosa by down-regulating the Toll-like receptor signaling pathway. In conclusion, Δpep27 seems to alleviate TLR signaling and NLRP3 inflammasome activation to subsequently prevent AR.

Crystal Structure of the Pneumococcal Vancomycin-Resistance Response Regulator DNA-Binding Domain.

Vancomycin response regulator (VncR) is a pneumococcal response regulator of the VncRS two-component signal transduction system (TCS) of Streptococcus pneumoniae. VncRS regulates bacterial autolysis and vancomycin resistance. VncR contains two different functional domains, the N-terminal receiver domain and C-terminal effector domain. Here, we investigated VncR C-terminal DNA binding domain (VncRc) structure using a crystallization approach. Crystallization was performed using the micro-batch method. The crystals diffracted to a 1.964 Å resolution and belonged to space group P212121. The crystal unit-cell parameters were a = 25.71 Å, b = 52.97 Å, and c = 60.61 Å. The structure of VncRc had a helix-turn-helix motif highly similar to the response regulator PhoB of Escherichia coli. In isothermal titration calorimetry and size exclusion chromatography results, VncR formed a complex with VncS, a sensor histidine kinase of pneumococcal TCS. Determination of VncR structure will provide insight into the mechanism by how VncR binds to target genes.

Corona-Cov-2 (COVID-19) and ginseng: Comparison of possible use in COVID-19 and influenza.

In the 1918 influenza pandemic, more than 95% of mortalities were ascribed to bacterial pneumonia. After the primary influenza infection, the innate immune system is attenuated, and the susceptibility to bacteria is increased. Subsequent bacterial pneumonia exacerbates morbidity and increases the mortality rate. Similarly, COVID-19 infection attenuates innate immunity and results in pneumonia. In addition, the current pneumococcal conjugate vaccine may have limited defense against secondary pneumococcal infection after influenza infection. Therefore, until a fully protective vaccine is available, a method of increasing immunity may be helpful. Ginseng has been shown to increase the defense against influenza in clinical trials and animal experiments, as well as the defense against pneumococcal pneumonia in animal experiments. Based on these findings, ginseng is suspected to be helpful for providing immunity against COVID-19.

ClpL is a functionally active tetradecameric AAA+ chaperone, distinct from hexameric/dodecameric ones.

AAA+ (ATPases associated with diverse cellular activities) chaperones are involved in a plethora of cellular activities to ensure protein homeostasis. The function of AAA+ chaperones is mostly modulated by their hexameric/dodecameric quaternary structures. Here we report the structural and biochemical characterizations of a tetradecameric AAA+ chaperone, ClpL from Streptococcus pneumoniae. ClpL exists as a tetradecamer in solution in the presence of ATP. The cryo-EM structure of ClpL at 4.5 Å resolution reveals a striking tetradecameric arrangement. Solution structures of ClpL derived from small-angle X-ray scattering data suggest that the tetradecameric ClpL could assume a spiral conformation found in active hexameric/dodecameric AAA+ chaperone structures. Vertical positioning of the middle domain accounts for the head-to-head arrangement of two heptameric rings. Biochemical activity assays with site-directed mutagenesis confirmed the critical roles of residues both in the integrity of the tetradecameric arrangement and activities of ClpL. Non-conserved Q321 and R670 are crucial in the heptameric ring assembly of ClpL. These results establish that ClpL is a functionally active tetradecamer, clearly distinct from hexameric/dodecameric AAA+ chaperones.

Korean Red Ginseng alleviates neuroinflammation and promotes cell survival in the intermittent heat stress-induced rat brain by suppressing oxidative stress via estrogen receptor beta and brain-derived neurotrophic factor upregulation.

BACKGROUND: Heat stress orchestrates neurodegenerative disorders and results in the formation of reactive oxygen species that leads to cell death. Although the immunomodulatory effects of ginseng are well studied, the mechanism by which ginseng alleviates heat stress in the brain remains elusive. METHODS: Rats were exposed to intermittent heat stress for 6 months, and brain samples were examined to elucidate survival and antiinflammatory effect after Korean Red Ginseng (KRG) treatment. RESULTS: Intermittent long-term heat stress (ILTHS) upregulated the expression of cyclooxygenase 2 and inducible nitric oxide synthase, increasing infiltration of inflammatory cells (hematoxylin and eosin staining) and the level of proinflammatory cytokines [tumor necrosis factor α, interferon gamma (IFN-γ), interleukin (IL)-1ß, IL-6], leading to cell death (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay) and elevated markers of oxidative stress damage (myeloperoxidase and malondialdehyde), resulting in the downregulation of antiapoptotic markers (Bcl-2 and Bcl-xL) and expression of estrogen receptor beta and brain-derived neurotrophic factor, key factors in regulating neuronal cell survival. In contrast, KRG mitigated ILTHS-induced release of proinflammatory mediators, upregulated the mRNA level of the antiinflammatory cytokine IL-10, and increased myeloperoxidase and malondialdehyde levels. In addition, KRG significantly decreased the expression of the proapoptotic marker (Bax), did not affect caspase-3 expression, but increased the expression of antiapoptotic markers (Bcl-2 and Bcl-xL). Furthermore, KRG significantly activated the expression of both estrogen receptor beta and brain-derived neurotrophic factor. CONCLUSION: ILTHS induced oxidative stress responses and inflammatory molecules, which can lead to impaired neurogenesis and ultimately neuronal death, whereas, KRG, being the antioxidant, inhibited neuronal damage and increased cell viability.

Ginseng alleviates microbial infections of the respiratory tract: a review.

The detrimental impact of air pollution as a result of frequent exposure to fine particles posed a global public health risk mainly to the pulmonary disorders in pediatric and geriatric population. Here, we reviewed the current literature regarding the role of ginseng and/or its components as antimicrobials, especially against pathogens that cause respiratory infections in animal and in vitro models. Some of the possible mechanisms for ginseng-mediated viral inhibition suggested are improvements in systemic and mucosa-specific antibody responses, serum hemagglutinin inhibition, lymphocyte proliferation, cell survival rate, and viral clearance in the lungs. In addition, ginseng reduces the expression levels of proinflammatory cytokines (IFN-γ, TNF-α, IL-2, IL-4, IL-5, IL-6, IL-8) and chemokines produced by airway epithelial cells and macrophages, thus preventing weight loss. In case of bacterial infections, ginseng acts by alleviating inflammatory cytokine production, increasing survival rates, and activating phagocytes and natural killer cells. In addition, ginseng inhibits biofilm formation and induces the dispersion and dissolution of mature biofilms. Most clinical trials revealed that ginseng, at various dosages, is a safe and effective method of seasonal prophylaxis, relieving the symptoms and reducing the risk and duration of colds and flu. Taken together, these findings support the efficacy of ginseng as a therapeutic and prophylactic agent for respiratory infections.

Pneumococcal VncR Strain-Specifically Regulates Capsule Polysaccharide Synthesis.

Capsular polysaccharides (CPS), a major virulence factor in Streptococcus pneumoniae, become thicker during blood invasion while not during asymptomatic nasopharyngeal colonization. However, the underlying mechanism controlling this differential pneumococcal CPS regulation remain unclear. Here, we show how VncR, the response regulator of the vancomycin resistance locus (vncRS operon), regulates CPS expression in vncR mutants in three serotype (type 2, 3, and 6B) backgrounds upon exposure to serum lactoferrin (LF). Comparative analysis of CPS levels in the wild type (WT) of three strains and their isogenic vncR mutants after LF exposure revealed a strain-specific alteration in CPS production. Consistently, VncR-mediated strain-specific CPS production is correlated with pneumococcal virulence, in vivo. Electrophoretic mobility-shift assay and co-immunoprecipitation revealed an interaction between VncR and the cps promoter (cpsp) in the presence of serum. In addition, in silico analysis uncovered this protein-DNA interaction, suggesting that VncR binds with the cpsp, and recognizes the strain-specific significance of the tandem repeats in cpsp. Taken together, the interaction of VncR and cpsp after serum exposure plays an essential role in regulating differential strain-specific CPS production, which subsequently determines strain-specific systemic virulence. This study highlights how host protein LF contributes to pneumococcal VncR-mediated CPS production. As CPS plays a significant role in immune evasion, these findings suggest that drugs designed to interrupt the VncR-mediated CPS production could help to combat pneumococcal infections.

Korean Red Ginseng enhances pneumococcal Δpep27 vaccine efficacy by inhibiting reactive oxygen species production.

BACKGROUND: Streptococcus pneumoniae, more than 90 serotypes of which exist, is recognized as an etiologic agent of pneumonia, meningitis, and sepsis associated with significant morbidity and mortality worldwide. Immunization with a pneumococcal pep27 mutant (Δpep27) has been shown to confer comprehensive, long-term protection against even nontypeable strains. However, Δpep27 is effective as a vaccine only after at least three rounds of immunization. Therefore, treatments capable of enhancing the efficiency of Δpep27 immunization should be identified without delay. Panax ginseng Mayer has already been shown to have pharmacological and antioxidant effects. Here, the ability of Korean Red Ginseng (KRG) to enhance the efficacy of Δpep27 immunization was investigated. METHODS: Mice were treated with KRG and immunized with Δpep27 before infection with the pathogenic S. pneumoniae strain D39. Total reactive oxygen species production was measured using lung homogenates, and inducible nitric oxide (NO) synthase and antiapoptotic protein expression was determined by immunoblotting. The phagocytic activity of peritoneal macrophages was also tested after KRG treatment. RESULTS: Compared with the other treatments, KRG significantly increased survival rate after lethal challenge and resulted in faster bacterial clearance via increased phagocytosis. Moreover, KRG enhanced Δpep27 vaccine efficacy by inhibiting reactive oxygen species production, reducing extracellular signal-regulated kinase apoptosis signaling and inflammation. CONCLUSION: Taken together, our results suggest that KRG reduces the time required for immunization with the Δpep27 vaccine by enhancing its efficacy.

Pneumococcal pep27 mutant immunization suppresses allergic asthma in mice.

Asthma is an allergic airway disease (AAD) characterized by eosinophilic inflammation, mucus hypersecretion, and airway hyper responsiveness, and it is caused by dysregulated immune responses. Conversely, regulatory T cells (Tregs) control aberrant immune responses and maintain homeostasis. Recent evidence suggests that Streptococcus pneumoniae, including its components as well as a live attenuated mutant, and pneumococcal infection induce Tregs and can thus potentially be harnessed therapeutically for asthma treatment. Previously, a pep27 deletion mutant (Δpep27) demonstrated a significantly attenuated virulence in a sepsis model, and Δpep27 immunization induced serotype-nonspecific protection against S. pneumoniae infection, as well as influenza virus, possibly via an immune tolerance mechanism. Here, the potential of Δpep27 immunization for asthma protection was studied. Mice were immunized intranasally with Δpep27 before or after ovalbumin sensitization and subsequent challenge. Δpep27 immunization suppressed hallmark features of AAD, including antigen-specific type 2 helper T cell cytokine and antibody responses, peripheral and pulmonary eosinophil accumulation, and goblet cell hyperplasia. Thus, a Δpep27 vaccine may be highly feasible as a preventive or therapeutic agent for asthma.

Sulfuretin Prevents Obesity and Metabolic Diseases in Diet Induced Obese Mice.

The global obesity epidemic and associated metabolic diseases require alternative biological targets for new therapeutic strategies. In this study, we show that a phytochemical sulfuretin suppressed adipocyte differentiation of preadipocytes and administration of sulfuretin to high fat diet-fed obese mice prevented obesity and increased insulin sensitivity. These effects were associated with a suppressed expression of inflammatory markers, induced expression of adiponectin, and increased levels of phosphorylated ERK and AKT. To elucidate the molecular mechanism of sulfuretin in adipocytes, we performed microarray analysis and identified activating transcription factor 3 (Atf3) as a sulfuretin-responsive gene. Sulfuretin elevated Atf3 mRNA and protein levels in white adipose tissue and adipocytes. Consistently, deficiency of Atf3 promoted lipid accumulation and the expression of adipocyte markers. Sulfuretin's but not resveratrol's anti-adipogenic effects were diminished in Atf3 deficient cells, indicating that Atf3 is an essential factor in the effects of sulfuretin. These results highlight the usefulness of sulfuretin as a new anti-obesity intervention for the prevention of obesity and its associated metabolic diseases.

Allicin induces beige-like adipocytes via KLF15 signal cascade.

Under specific conditions, white adipose tissue (WAT) depots are readily converted to a brown-like state, which is associated with weight loss. However, whether diet-derived factors directly induce browning of white adipocytes has yet to be established. Thus, we investigated the effects of allicin, one of the major components of garlic, on brown-like adipocyte formation in inguinal WAT (iWAT), and prevention of obesity and related complications in animal models. Allicin significantly increased mRNA and/or protein expression of brown adipocyte markers including uncoupling protein 1 (UCP1) in differentiated mouse embryonic fibroblast cell line 3T3-L1 and differentiated iWAT stromal vascular cells (SVC), suggesting that allicin induced brown-like adipocyte formation in vitro. Concomitantly, allicin markedly enhanced the protein expression of KLF-15 and its interaction with UCP-1 promoter region. Such changes were absent in cells lacking KLF-15, suggesting the critical role of KLF15 in allicin action. Allicin also induced brown-like adipogenesis in vivo along with the appearance of multilocular adipocytes, increased UCP1 expression and increased lipid oxidation. In summary, our data suggest that allicin potentially prevents obesity and associated metabolic disorders such as type 2 diabetes mellitus by enhancing the expression of brown adipocyte-specific genes, including UCP-1, through KLF15 signal cascade.

Immunization with attenuated non-transformable pneumococcal pep27 and comD mutant provides serotype-independent protection against pneumococcal infection.

Streptococcus pneumoniae is a well-known pathogenic bacterium with a high mortality rate. Currently, a 23-valent pneumococcal polysaccharide vaccine (PPV23) and protein-conjugate vaccines (PCVs) are available on the market. However, both of these vaccines have limitations; specifically, PPV23 produces weak antibody responses in children younger than 2 years and PCVs only partially protect against secondary infection. Previously, we showed serotype-nonspecific protection by Δpep27 vaccine, but the reversion of Δpep27 to the wild type serotype during immunization cannot be excluded. To ensure the safety of the Δpep27 vaccine, comD, an important protein that activates competence, was inactivated, and the transformability of the double mutant (Δpep27ΔcomD) was determined. The transformation ability of this double mutant was successfully abolished. Δpep27ΔcomD immunization significantly increased the survival time after heterologous challenge(s), and diminished colonization levels independent of serotype, including a non-typeable strain (NCC1). Moreover, the double mutant was found to be highly safe in both normal and immunocompromised mice. In conclusion, this pneumococcal Δpep27ΔcomD vaccine appears to be a highly feasible and safe vaccine to prevent various types of pneumococcal infections.

Induction of the pneumococcal vncRS operon by lactoferrin is essential for pneumonia.

Streptococcus pneumoniae (pneumococcus), the major pathogen for pneumonia, commonly colonizes the lung, but the mechanism underlying the coordination of virulence factors during invasion via the host protein remains poorly understood. Bacterial lysis releases the components of the cell wall, and triggers innate immunity and the subsequent secretion of pro-inflammatory cytokines. Previously, the virulence of the pep27 mutant was shown to be attenuated as a feasible candidate for vaccine development. However, the role of pep27 gene, belonging to the vancomycin-resistance locus (vncRS operon), in virulence, is largely unknown. This study demonstrates that transferrin in the host serum reduces the survival of the host during S. pneumoniae infections in mice. The exposure of the pneumococcal D39 strain to lactoferrin induced the vncRS operon, lysis, and subsequent in vivo cytokine production, resulting in lung inflammation. However, these responses were significantly attenuated in pneumococci harboring a mutation in pep27. Mechanistically, the VncS ligand, identified as lactoferrin, induced the vncRS operon and increased the in vivo mortality rates. Thus, serum-induced activation of vncRS plays an essential role in inducing pneumonia.