Aerobic physical training reduces severe asthma phenotype involving kinins pathway.

INTRODUCTION: Aerobic physical training (APT) reduces eosinophilic airway inflammation, but its effects and mechanisms in severe asthma remain unknown. METHODS: An in vitro study employing key cells involved in the pathogenesis of severe asthma, such as freshly isolated human eosinophils, neutrophils, and bronchial epithelial cell lineage (BEAS-2B) and lung fibroblasts (MRC-5 cells), was conducted. Additionally, an in vivo study using male C57Bl/6 mice, including Control (Co; n = 10), Trained (Exe; n = 10), house dust mite (HDM; n = 10), and HDM + Trained (HDM + Exe; n = 10) groups, was carried out, with APT performed at moderate intensity, 5x/week, for 4 weeks. RESULTS: HDM and bradykinin, either alone or in combination, induced hyperactivation in human neutrophils, eosinophils, BEAS-2B, and MRC-5 cells. In contrast, IL-10, the primary anti-inflammatory molecule released during APT, inhibited these inflammatory effects, as evidenced by the suppression of numerous cytokines and reduced mRNA expression of the B1 receptor and ACE-2. The in vivo study demonstrated that APT decreased bronchoalveolar lavage levels of bradykinin, IL-1ß, IL-4, IL-5, IL-17, IL-33, TNF-α, and IL-13, while increasing levels of IL-10, klotho, and IL-1RA. APT reduced the accumulation of polymorphonuclear cells, lymphocytes, and macrophages in the peribronchial space, as well as collagen fiber accumulation, epithelial thickness, and mucus accumulation. Furthermore, APT lowered the expression of the B1 receptor and ACE-2 in lung tissue and reduced bradykinin levels in the lung tissue homogenate compared to the HDM group. It also improved airway resistance, tissue resistance, and tissue damping. On a systemic level, APT reduced total leukocytes, eosinophils, neutrophils, basophils, lymphocytes, and monocytes in the blood, as well as plasma levels of IL-1ß, IL-4, IL-5, IL-17, TNF-α, and IL-33, while elevating the levels of IL-10 and IL-1RA. CONCLUSION: These findings indicate that APT inhibits the severe asthma phenotype by targeting kinin signaling.

Vitamin C Inhibits Lipopolysaccharide-Induced Hyperinflammatory State of Chronic Myeloid Leukemia Cells through Purinergic Signaling and Autophagy.

Background: Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm characterized by the overproduction of white blood cells, leading to symptoms such as fatigue, infections, and other complications. CML patients must take measures to prevent infections to mitigate the exacerbation of cancer cell proliferation and comorbidities. Methods: This study investigated whether vitamin C can suppress the hyperinflammatory activation of K-562 cells induced by lipopolysaccharide (LPS) and whether purinergic signaling (ATP and P2X7 receptor) and autophagy play a role in it. Two different doses of vitamin C (5 µg/mL and 10 µg/mL) were employed, along with the lysosome inhibitor chloroquine (CQ; 100 µM), administered 2 h prior to LPS stimulation (10 ng/mL) for a duration of 22 h in K-562 cells (3 × 105 cells/mL/well). Results: Both doses of vitamin C reduced the release of interleukin-6 (IL-6) (5 µg/mL, p < 0.01 and 10 µg/mL, p < 0.01) and tumor necrosis factor (TNF) (5 µg/mL, p < 0.01 and 10 µg/mL, p < 0.01) induced by LPS. Furthermore, in LPS + CQ-stimulated cells, vitamin C at a concentration of 10 µg/mL inhibited the expression of LC3-II (p < 0.05). Conversely, both doses of vitamin C led to the release of the anti-inflammatory cytokine interleukin-10 (IL-10) (5 µg/mL, p < 0.01 and 10 µg/mL, p < 0.01), while only the 10 µg/mL dose of vitamin C induced the release of Klotho (10 µg/mL, p < 0.01). In addition, both doses of vitamin C reduced the accumulation of ATP (5 µg/mL, p < 0.01 and 10 µg/mL, p < 0.01) and decreased the expression of the P2X7 receptor at the mRNA level. Conclusions: Vitamin C inhibits the hyperinflammatory state induced by LPS in K-562 cells, primarily by inhibiting the ATP accumulation, P2X7 receptor expression, and autophagy signaling.

Lactobacillus rhamnosus Modulates Lung Inflammation and Mitigates Gut Dysbiosis in a Murine Model of Asthma-COPD Overlap Syndrome.

The asthma-COPD overlap syndrome (ACOS) presents lung inflammation similar to both asthma and chronic obstructive pulmonary disease (COPD). Due to the immune response between the lung and gut, it is possible that ACOS individuals present gut dysbiosis. Due to therapeutic limitations in ACOS, Lactobacillus rhamnosus (Lr) have received attention once Lr has been effective in asthma and COPD. However, there is no data about the Lr effect on both lung inflammation and gut dysbiosis in ACOS. Thus, our study investigated the Lr effect on lung inflammation, bronchoconstriction, airway remodeling, and gut dysbiosis in the murine ACOS model. Treated mice with Lr were exposed to HDM and cigarette smoke to induce ACOS. Sixty days after ACOS induction, mice were euthanized. Lung inflammation was evaluated in leukocytes in bronchoalveolar lavage fluid (BALF), airway remodeling, cytokine secretion, and transcription factor expression in the lung. The gut microbiota was assayed by 16S mRNA sequencing from a fecal sample. Leukocyte population, bronchial hyperreactivity, pro-inflammatory cytokines, and airway remodeling were attenuated in Lr-treated ACOS mice. Likewise, IL-4, IL-5, and IL-13, STAT6 and GATA3, as well as IL-17, IL-21, IL-22, STAT3, and RORÉ£t were reduced after Lr. In addition, IL-2, IL-12, IFN-γ, STAT1, and T-bet as well as IL-10, TGF-ß, STAT5, and Foxp3 were restored after the Lr. Firmicutes was reduced, while Deferribacteres was increased after Lr. Likewise, Lr decreased Staphylococcus and increased Mucispirillum in ACOS mice. Lr improves fecal bacterial ß-diversity. Our findings show for the first time the Lr effect on lung inflammation and gut dysbiosis in murine ACOS.

Lactobacillus rhamnosus Restores Antiviral Signaling and Attenuates Cytokines Secretion from Human Bronchial Epithelial Cells Exposed to Cigarette Smoke and Infected with SARS-CoV-2.

Individuals with chronic obstructive pulmonary disease (COPD) are more susceptible to exacerbation crisis triggered by secondary lung infections due to the dysfunction of antiviral signaling, principally via suppression of IFN-γ. Although the probiotic is known for controlling pulmonary inflammation in COPD, the influence of the Lactobacillus rhamnosus (Lr) on antiviral signaling in bronchial epithelium exposed to cigarette smoke extract (CSE) and viruses, remains unknown. Thus, the present study investigated the Lr effect on the antiviral signaling and the secretion of inflammatory mediators from bronchial epithelial cells (16HBE cells) exposed to CSE and SARS-CoV-2. The 16HBE cells were cultured, treated with Lr, stimulated with CSE, and infected with SARS-CoV-2. The cellular viability was evaluated using the MTT assay and cytotoxicity measured by lactate dehydrogenase (LDH) activity. The viral load, TLR2, TLR3, TLR4, TLR7, TLR8, MAVS, MyD88, and TRIF were quantified using specific PCR. The pro-inflammatory mediators were measured by a multiplex biometric immunoassay, and angiotensin converting enzyme 2 (ACE2) activity, NF-κB, RIG-I, MAD5, and IRF3 were measured using specific ELISA kits. Lr decreased viral load, ACE2, pro-inflammatory mediators, TLR2, TLR4, NF-κB, TLR3, TLR7, and TLR8 as well as TRIF and MyD88 expression in CSE and SARS-CoV-2 -exposed 16HBE cells. Otherwise, RIG-I, MAD5, IRF3, IFN-γ, and the MAVS expression were restored in 16HBE cells exposed to CSE and SARS-CoV-2 and treated with Lr. Lr induces antiviral signaling associated to IFN-γ secreting viral sensors and attenuates cytokine storm associated to NF-κB in bronchial epithelial cells, supporting its emerging role in prevention of COPD exacerbation.

A Nutritional Blend Suppresses the Inflammatory Response from Bronchial Epithelial Cells Induced by SARS-CoV-2.

Even after virus elimination, numerous sequelae of coronavirus disease 2019 (COVID-19) persist. Based on accumulating evidence, large amounts of proinflammatory cytokines are released to drive COVID-19 progression, severity, and mortality, and their levels remain elevated after the acute phase of COVID-19, playing a central role in the disease' sequelae. In this manner, bronchial epithelial cells are the first cells hyperactivated by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), leading to massive cytokine release, triggering the hyperactivation of leukocytes and other cells, and mediating COVID-19 sequelae. Therefore, proinflammatory cytokine production is initiated by the host. This in vitro study tested the hypothesis that ImmuneRecov™, a nutritional blend, inhibits the SARS-CoV-2-induced hyperactivation of human bronchial epithelial cells (BEAS-2B). BEAS-2B (5x104/mL/well) cells were cocultivated with 1 ml of blood from a SARS-CoV-2-infected patient for 4 h, and the nutritional blend (1 µg/mL) was added in the first minute of coculture. After 4 h, the cells were recovered and used for analyses of cytotoxicity with the (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) (MTT) assay and the expression of the IL-1ß, IL-6, and IL-10 mRNAs. The supernatant was collected to measure cytokine levels. SARS-CoV-2 incubation resulted in increased levels of IL-1ß and IL-6 in BEAS-2B cells (p < 0.001). Treatment with the nutritional blend resulted in reduced levels of the proinflammatory cytokines IL-1ß and IL-6 (p < 0.001) and increased levels of the anti-inflammatory cytokine IL-10 (p < 0.001). Additionally, the nutritional blend reduced the expression of the IL-1ß and IL-6 mRNAs in SARS-CoV-2-stimulated cells and increased the expression of the IL-10 and IFN-γ mRNAs. In conclusion, the nutritional blend exerts important anti-inflammatory effects on cells in the context of SARS-CoV-2 infection.

Hydrolyzed Collagen Induces an Anti-Inflammatory Response That Induces Proliferation of Skin Fibroblast and Keratinocytes.

Collagen-based products are found in different pharmaceuticals, medicine, food, and cosmetics products for a wide variety of applications. However, its use to prevent or improve the health of skin is growing dizzyingly. Therefore, this study investigated whether collagen peptides could induce fibroblast and keratinocyte proliferation and activation beyond reducing an inflammatory response induced by lipopolysaccharide (LPS). Human skin fibroblasts (CCD-1072Sk) and human keratinocytes (hKT-nh-skp-KT0026) were seeded at a concentration of 5 × 104 cells/mL. LPS (10 ng/mL) and three doses of collagen peptides (2.5 mg/mL, 5 mg/mL, 10 mg/mL) were used. The readout parameters were cell proliferation; expression of inducible nitric oxide synthase (iNOS); expression of pro-collagen-1α by fibroblasts; and secretion of interleukin-1ß (IL-1ß), interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor α (TNF-α), transforming growth factor ß (TGF-ß), and vascular endothelial growth factor (VEGF) by both cell types. The results demonstrated that all doses of collagen supplementation induced increased proliferation of both human fibroblasts (p < 0.01) and human keratinocytes (p < 0.001), while only the dose of 10 mg/mL induced an increased expression of pro-collagen-1α by fibroblasts. Similarly, only the dose of 10 mg/mL reduced LPS-induced iNOS expression in fibroblasts (p < 0.05) and keratinocytes (p < 0.01). In addition, collagen supplementation reduced the LPS-induced IL-1ß (p < 0.05), IL-6 (p < 0.001), IL-8 (p < 0.01), and TNF-α (p < 0.05), and increased the TGF-ß and VEGF expression in fibroblasts. Furthermore, collagen supplementation reduced the LPS-induced IL-1ß (p < 0.01), IL-6 (p < 0.01), IL-8 (p < 0.01), and TNF-α (p < 0.001), and increased the TGF-ß (p < 0.05) and VEGF (p < 0.05) expression in keratinocytes. In conclusion, collagen peptides were found to induce fibroblast and keratinocyte proliferation and pro-collagen-1α expression, involving increased expression of TGF-ß and VEGF, as well as the suppression of an inflammatory response induced by LPS.

Lacticaseibacillus rhamnosus modulates the inflammatory response and the subsequent lung damage in a murine model of acute lung inflammation.

OBJECTIVE: The present study investigated the anti-inflammatory effect of the probiotic Lacticaseibacillus rhamnosus (Lr) on lung inflammation induced by Lipopolysaccharide (LPS) of Escherichia coli in C57BL/6 mice. METHODS: C57BL/6 mice were divided into four groups: control, LPS, Lr (1 day) + LPS, and Lr (14 days) + LPS. Total and differential cells from Bronchoalveolar Lavage Fluid (BALF) were counted in a Neubauer 40X chamber, and pro-and anti-inflammatory cytokines (IL-1ß, IL-6, CXCL-1, TNF-α, TGF-ß, and IL-10) were measured by ELISA assay. The analysis of whole leukocytes in blood was performed using the automated system Sysmex 800i. Morphometry of pulmonary tissue evaluated alveolar hemorrhage, alveolar collapse, and inflammatory cells. Pulmonary vascular permeability was assessed by Evans blue dye extravasation, and bronchoconstriction was evaluated in a tissue bath station. The transcription factor NF-kB was evaluated by ELISA, and its gene expression and TLR-2, TLR-4, MMP-9, MMP-12, and TIMP by PCR. RESULTS: The probiotic Lr had a protective effect against the inflammatory responses induced by LPS. Lr significantly reduced pro-inflammatory cells in the airways, lung parenchyma, and blood leukocytes. Furthermore, Lr reduced the production of pro-inflammatory cytokines and chemokines in BALF and the expression of TLRs, MMPs, and NF-kB in lung tissue and maintained the expression of TIMP in treated animals promoting a protective effect on lung tissue. CONCLUSIONS: The results of the study indicate that pre-treatment with the probiotic Lr may be a promising way to mitigate lung inflammation in endotoxemia.

Transfer factors peptides (Imuno TF®) modulate the lung inflammation and airway remodeling in allergic asthma.

Background: Allergic asthma is a chronic lung disease in which the lung inflammation and airway remodeling are orchestrated by both the inflammatory and the immune cells that creates a lung millieu that favors the perpetuation of clinical symptoms. The cell signaling in asthma involves the mast cells activation during initial contact with the allergen and, principally, the participation of eosinophils as well as Th2 cells which determine increased levels of IgE, exaggerated secretion of mucus and collagen, and bronchial hyperreactivity. Moreover, allergic asthma presents lower level of cytokines associated to the both Th1 and Treg cells response, and it implies in deficiency of anti-inflammatory response to counterregulate the exaggerated inflammation against allergen. Therefore, the equilibrium between cytokines as well as transcription factors associated to Th2, Th1, and Treg cells is compromised in allergic asthma. Imuno TF® is a food supplement with ability to interfere in immune system pathways. It has been previously demonstrated that Imuno TF® upregulated Th1 cell response whilst downregulated Th2 cell response in human lymphocytes. Objective: For this reason, we hypothesized that the Imuno TF effect could be restore the balance between Th1/Th2 CD4 T cells response in murine allergic asthma. Methods: Initially, animals were sensitized with OVA via i.p. and challenged with OVA i.n. on days 14, 15 and 16. Treatment with Imuno TF once a day was performed via orogastric from day 17 to day 20. Mice were euthanized on day 21. Results: The Imuno TF reduced eosinophilia, mucus production, and airway remodeling (collagen deposition) in asthma mice. Imuno TF influenced cellular signaling associated to allergic asthma once downregulated STAT6 expression as well as decreased IL-4, IL-5, and IL-13 in lung and serum. In addition, Imuno TF restored T-bet and Foxp3 expression as well as increased IL-12, IFN-É£, and IL-10. Conclusion: Ultimately, Imuno TF mitigated the allergic asthma due to the restoration of balance between the responses of Th1/Th2 as well as Treg cells, and their respective transcription factors the T-bet/STAT6 and Foxp3.

Lacticaseibacillus rhamnosus modulates the inflammatory response and the subsequent lung damage in a murine model of acute lung inflammation

ABSTRACT Objective: The present study investigated the anti-inflammatory effect of the probiotic Lacticaseibacillus rhamnosus (Lr) on lung inflammation induced by Lipopolysaccharide (LPS) of Escherichia coli in C57BL/6 mice. Methods: C57BL/6 mice were divided into four groups: control, LPS, Lr (1 day) + LPS, and Lr (14 days) + LPS. Total and differential cells from Bronchoalveolar Lavage Fluid (BALF) were counted in a Neubauer 40X chamber, and pro-and anti-inflammatory cytokines (IL-1β, IL-6, CXCL-1, TNF-α, TGF-β, and IL-10) were measured by ELISA assay. The analysis of whole leukocytes in blood was performed using the automated system Sysmex 800i. Morphometry of pulmonary tissue evaluated alveolar hemorrhage, alveolar collapse, and inflammatory cells. Pulmonary vascular permeability was assessed by Evans blue dye extravasation, and bronchoconstriction was evaluated in a tissue bath station. The transcription factor NF-kB was evaluated by ELISA, and its gene expression and TLR-2, TLR-4, MMP-9, MMP-12, and TIMP by PCR. Results: The probiotic Lr had a protective effect against the inflammatory responses induced by LPS. Lr significantly reduced pro-inflammatory cells in the airways, lung parenchyma, and blood leukocytes. Furthermore, Lr reduced the production of pro-inflammatory cytokines and chemokines in BALF and the expression of TLRs, MMPs, and NF-kB in lung tissue and maintained the expression of TIMP in treated animals promoting a protective effect on lung tissue. Conclusions: The results of the study indicate that pre-treatment with the probiotic Lr may be a promising way to mitigate lung inflammation in endotoxemia.