Characterization of a Cutibacterium acnes Camp Factor 1-Related Peptide as a New TLR-2 Modulator in In Vitro and Ex Vivo Models of Inflammation

Cutibacterium acnes (C. acnes) has been implicated in inflammatory acne where highly mutated Christie–Atkins–Munch–Petersen factor (CAMP)1 displays strong toll like receptor (TLR)-2 binding activity. Using specific antibodies, we showed that CAMP1 production was independent of C. acnes phylotype and involved in the induction of inflammation. We confirmed that TLR-2 bound both mutated and non-mutated recombinant CAMP1, and peptide array analysis showed that seven peptides (A14, A15, B1, B2, B3, C1 and C3) were involved in TLR-2 binding, located on the same side of the three-dimensional structure of CAMP1. Both mutated and non-mutated recombinant CAMP1 proteins induced the production of C-X-C motif chemokine ligand interleukin (CXCL)8/(IL)-8 in vitro in keratinocytes and that of granulocyte macrophage-colony stimulating factor (GM-CSF), tumor necrosis factor (TNF)-α, IL-1β and IL-10 in ex vivo human skin explants. Only A14, B1 and B2 inhibited the production of CXCL8/IL-8 by keratinocytes and that of (GM-CSF), TNF-α, IL-1β and IL-10 in human skin explants stimulated with rCAMP1 and C. acnes. Following pretreatment with B2, RNA sequencing on skin explants identified the 10 genes displaying the strongest differential expression as IL6, TNF, CXCL1, CXCL2, CXCL3, CXCL8, IL-1β, chemokine ligand (CCL)2, CCL4 and colony stimulating factor (CSF)2. We, thus, identified a new CAMP1-derived peptide as a TLR-2 modulator likely to be a good candidate for clinical evaluation.     

A20 Overexpression Inhibits Lipopolysaccharide-Induced NF-κB Activation,TRAF6 and CD40 Expression in Rat Peritoneal Mesothelial Cells

Zinc finger protein A20 is a key negative regulator of inflammation. However, whether A20 may affect inflammation during peritoneal dialysis (PD)-associated peritonitis is still unclear. This study was aimed to investigate the effect of A20 overexpression on lipopolysaccharide (LPS)-induced inflammatory response in rat peritoneal mesothelial cells (RPMCs). Isolated and cultured RPMCs in vitro. Plasmid pGEM-T easy-A20 was transfected into RPMCs by Lipofectamine™2000. The protein expression of A20, phospho-IκBα, IκBα, TNF receptor-associated factor (TRAF) 6 and CD40 were analyzed by Western blot. The mRNA expression of TRAF6, CD40, interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were determined by real time-PCR. NF-κB p65 DNA binding activity, IL-6 and TNF-α levels in cells culture supernatant were determined by ELISA. Our results revealed that RPMCs overexpression of A20 lead to significant decrease of LPS-induced IκBα phosphorylation and NF-κB DNA binding activity (all p < 0.01). In addition, A20 also attenuated the expression of TRAF6, CD40, IL-6 and TNF-α as well as levels of IL-6 and TNF-α in cells culture supernatant (all p < 0.05). However, A20 only partly inhibited CD40 expression. Our study indicated that A20 overexpression may depress the inflammatory response induced by LPS in cultured RPMCs through negatively regulated the relevant function of adaptors in LPS signaling pathway.     

Methyl Brevifolincarboxylate Attenuates Free Fatty Acid-Induced Lipid Metabolism and Inflammation in Hepatocytes through AMPK/NF-κB Signaling Pathway

The prevalence of non-alcoholic fatty liver disease (NAFLD) is one of the leading causes of chronic liver diseases worldwide. This study examined the potential protective effects of a naturally occurring polyphenolic compound, methyl brevifolincarboxylate (MBC) on fatty liver injury in vitro. The results showed that MBC at its non-cytotoxic concentrations, reduced lipid droplet accumulation and triglyceride (TG) levels in the oleic acid (OA)-treated human hepatocarcinoma cell line, SK-HEP-1 and murine primary hepatocytes. In OA-treated SK-HEP-1 cells and primary murine hepatocytes, MBC attenuated the mRNA expression levels of the de novo lipogenesis molecules, acetyl-coenzyme A carboxylase (Acc1), fatty acid synthase (Fasn) and sterol regulatory element binding protein 1c (Srebp1c). MBC promoted the lipid oxidation factor peroxisome proliferator activated receptor-α (Pparα), and its target genes, carnitine palmitoyl transferase 1 (Cpt1) and acyl-coenzyme A oxidase 1 (Acox1) in both the SK-HEP-1 cells and primary murine hepatocytes. The mRNA results were further supported by the attenuated protein expression of lipogenesis and lipid oxidation molecules in OA-treated SK-HEP-1 cells. The MBC increased the expression of AMP activated protein kinase (AMPK) phosphorylation. On the other hand, MBC treatment dampened the inflammatory mediator’s, tumor necrosis factor (TNF)-α, interleukin-6 (IL-6), IL-8, and IL-1β secretion, and nuclear factor (NF)-κB expression (mRNA and protein) through reduced reactive oxygen species production in OA-treated SK-HEP-1 cells. Taken together, our results demonstrated that MBC possessed potential protective effects against NAFLD in vitro by amelioration of lipid metabolism and inflammatory markers through the AMPK/NF-κB signaling pathway.     

Deficit in Adipose Differentiation in Mesenchymal Stem Cells Derived from Chronic Rhinosinusitis Nasal Polyps Compared to Nasal Mucosal Tissue

Chronic rhinosinusitis of the nasal mucosa is an inflammatory disease of paranasal sinuses, which causes rhinorrhea, nasal congestion, and hyposmia, and in some cases, it can result in the development of nasal polyposis. Nasal polyps are benign lobular-shaped growths that project in the nasal cavities; they originate from inflammation in the paranasal mucous membrane and are associated with a high expression of interleukins (IL)-4, IL-5, IL-13, and IgE. Polyps derive from the epithelial–mesenchymal transition of the nasal epithelium resulting in a nasal tissue remodeling. Nasal polyps from three patients with chronic rhinosinusitis as well as control non-polyp nasal mucosa were used to isolate and cultivate mesenchymal stem cells characterized as CD73⁺, CD90⁺, CD105⁺/CD14⁻, CD34⁻, and CD45⁻. Mesenchymal stem cells (MSCs) cultures were induced to differentiate toward adipocytes, where lipid droplets and adipocyte genes PPARγ2, ADIPO-Q, and FABP4 were observed in control non-polyp nasal mucosa-derived mesenchymal cells but were scarcely present in the cultures derived from the nasal polyps, where apoptosis was evident. The modulation of the response to adipogenic stimulus in polyps represents a change in the molecular response that controls the cascade required for differentiation as well as possible means to specifically target these cells, sparing the normal mucosa of the nasal sinuses.     

Assessment of Blood Flow in Hepatocellular Carcinoma: Correlations of Computed Tomography Perfusion Imaging and Circulating Angiogenic Factors

Hepatocellular carcinoma (HCC) is a highly vascular tumor through the process of angiogenesis. To evaluate more non-invasive techniques for assessment of blood flow (BF) in HCC, this study examined the relationships between BF of HCC measured by computer tomography (CT) perfusion imaging and four circulating angiogenic factors in HCC patients. Interleukin 6 (IL-6), interleukin 8 (IL-8), vascular endothelial growth factor (VEGF), and platelet derived growth factor (PDGF) in plasma were measured using Bio-Plex multiplex immunoassay in 21 HCC patients and eight healthy controls. Circulating IL-6, IL-8 and VEGF showed higher concentrations in HCC patients than in controls (p < 0.05), and predicted HCC occurrence better than chance (p < 0.01). Twenty-one patients with HCC received 21-phase liver imaging using a 64-slice CT. Total BF, arterial BF, portal BF, arterial fraction (arterial BF/total BF) of the HCC and surrounding liver parenchyma, and HCC-parenchyma ratio were measured using a dual-vessel model. After analyzing the correlations between BF in HCC and four circulating angiogenic factors, we found that the HCC-parenchyma ratio of arterial BF showed a significantly positive correlation with the level of circulating IL-8 (p < 0.05). This circulating biomarker, IL-8, provides a non-invasive tool for assessment of BF in HCC.     

Immunosenescence in Aging-Related Vascular Dysfunction

The immunosenescence-related disproportion in T lymphocytes may have important consequences for endothelial dysfunction, which is a key event in vascular aging. The study was designed to assess the prognostic values of the inflammatory-immune profile to better predict and prevent vascular diseases associated with old age. Eighty individuals aged 70.9 ± 5.3 years were allocated to a low- (LGI) or high-grade inflammation (HGI) group based on CRP (<3 or ≥3 mg/L) as a conventional risk marker of cardiovascular diseases. Significant changes in inflammatory and endothelium-specific variables IL-1β, IL-6, TNFα, oxLDL, H₂O₂, NO, 3-nitrotyrosine, and endothelial progenitor cells (OR 7.61, 95% CI 2.56–29.05, p < 0.0001), confirmed their interplay in vascular inflammation. The flow-cytometry analysis demonstrated a high disproportion in T lymphocytes CD4⁺ and CD8⁺ between LGI and HGI groups. CRP was <3 mg/mL for the CD4/CD8 ratio within the reference values ≥ 1 or ≤2.5, unlike for the CD4/CD8 ratio < 1 and >2.5. The odds ratios for the distribution of CD4⁺ (OR 5.98, 95% CI 0.001–0.008, p < 0.001), CD8⁺ (OR 0.23, 95% CI 0.08–0.59, p < 0.01), and CD8CD45RO⁺ T naïve cells (OR 0.27, 95% CI 0.097–0.695, p < 0.01) and CD4/CD8 (OR 5.69, 95% CI 2.07–17.32, p < 0.001) indicated a potential diagnostic value of T lymphocytes for clinical prognosis in aging-related vascular dysfunction.     

Suppression of Cutibacterium acnes-Mediated Inflammatory Reactions by Fibroblast Growth Factor 21 in Skin

Cutibacterium acnes (C. acnes) is a common commensal bacterium that is closely associated with the pathogenesis of acne. Fibroblast growth factor 21 (FGF21), as a favorable regulator of glucose and lipid metabolism and insulin sensitivity, was recently shown to exert anti-inflammatory effects. The role and mechanism of FGF21 in the inflammatory reactions induced by C. acnes, however, have not been determined. The present study shows that FGF21 in the dermis inhibits epidermal C. acnes-induced inflammation in a paracrine manner while it functions on the epidermal layer through a receptor complex consisting of FGF receptor 1 (FGFR1) and β-Klotho (KLB). The effects of FGF21 in heat-killed C. acnes-induced HaCaT cells and living C. acnes-injected mouse ears were examined. In the presence of C. acnes, FGF21 largely counteracted the activation of Toll-like receptor 2 (TLR2), the downstream nuclear factor-κB (NF-κB), and mitogen-activated protein kinase (MAPK) signaling pathways induced by C. acnes. FGF21 also significantly reduced the expression of proinflammatory cytokines, including interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor (TNF)-α. Taken together, these findings indicate that FGF21 suppresses C. acnes-induced inflammation and might be used clinically in the management and treatment of acne.     

A Clearance Period after Soluble Lead Nanoparticle Inhalation Did Not Ameliorate the Negative Effects on Target Tissues Due to Decreased Immune Response

The inhalation of metal (including lead) nanoparticles poses a real health issue to people and animals living in polluted and/or industrial areas. In this study, we exposed mice to lead(II) nitrate nanoparticles [Pb(NO₃)₂ NPs], which represent a highly soluble form of lead, by inhalation. We aimed to uncover the effects of their exposure on individual target organs and to reveal potential variability in the lead clearance. We examined (i) lead biodistribution in target organs using laser ablation and inductively coupled plasma mass spectrometry (LA-ICP-MS) and atomic absorption spectrometry (AAS), (ii) lead effect on histopathological changes and immune cells response in secondary target organs and (iii) the clearance ability of target organs. In the lungs and liver, Pb(NO₃)₂ NP inhalation induced serious structural changes and their damage was present even after a 5-week clearance period despite the lead having been almost completely eliminated from the tissues. The numbers of macrophages significantly decreased after 11-week Pb(NO₃)₂ NP inhalation; conversely, abundance of alpha-smooth muscle actin (α-SMA)-positive cells, which are responsible for augmented collagen production, increased in both tissues. Moreover, the expression of nuclear factor κB (NF-κB) and selected cytokines, such as tumor necrosis factor alpha (TNFα), transforming growth factor beta 1 (TGFβ1), interleukin 6(IL-6), IL-1α and IL-1β , displayed a tissue-specific response to lead exposure. In summary, diminished inflammatory response in tissues after Pb(NO₃)₂ NPs inhalation was associated with prolonged negative effect of lead on tissues, as demonstrated by sustained pathological changes in target organs, even after long clearance period.     

Regulatory T Cells Exhibit Interleukin-33-Dependent Migratory Behavior during Skin Barrier Disruption

Regulatory T cells (Tregs) suppress immune responses and maintain immunological self-tolerance and homeostasis. We currently investigated relationships between skin barrier condition and Treg behavior using skin barrier-disrupted mice. Skin barrier disruption was induced by repeated topical application of 4% sodium dodecyl sulfate (SDS) on mice. The number of CD4⁺ forkhead box protein P3 (Foxp3)⁺ Tregs was higher in 4% SDS-treated skins than in controls. This increasing was correlated with the degree of acanthosis. The numbers of interleukin (IL)-10⁺ and transforming growth factor (TGF)-β⁺ Tregs also increased in 4% SDS-treated skins. Localization of IL-33 in keratinocytes shifted from nucleus to cytoplasm after skin barrier disruption. Notably, IL-33 promoted the migration of Tregs in chemotaxis assay. The skin infiltration of Tregs was cancelled in IL-33 neutralizing antibody-treated mice and IL-33 knockout mice. Thus, keratinocyte-derived IL-33 may induce Treg migration into barrier-disrupted skin to control the phase transition between healthy and inflammatory conditions.     

Menstrual Blood-Derived Endometrial Stem Cells’ Impact for the Treatment Perspective of Female Infertility

When looking for the causes and treatments of infertility, much attention is paid to one of the reproductive tissues—the endometrium. Therefore, endometrial stem cells are an attractive target for infertility studies in women of unexplained origin. Menstrual blood stem cells (MenSCs) are morphologically and functionally similar to cells derived directly from the endometrium; with dual expression of mesenchymal and embryonic cell markers, they proliferate and regenerate better than bone marrow mesenchymal stem cells. In addition, menstrual blood stem cells are extracted in a non-invasive and painless manner. In our study, we analyzed the characteristics and the potential for decidualization of menstrual blood stem cells isolated from healthy volunteers and women diagnosed with infertility. We demonstrated that MenSCs express CD44, CD166, CD16, CD15, BMSC, CD56, CD13 and HLA-ABC surface markers, have proliferative properties, and after induction of menstrual stem cell differentiation into epithelial direction, expression of genes related to decidualization (PRL, ESR, IGFBP and FOXO1) and angiogenesis (HIF1, VEGFR2 and VEGFR3) increased. Additionally, the p53, p21, H3K27me3 and HyperAcH4 proteins’ expression increased during MenSCs decidualization, they secrete proteins that are involved in the regulation of the actin cytoskeleton, estrogen and relaxin signaling pathways and the management of inflammatory processes. Our findings reveal the potential use of MenSCs for the treatment of reproductive disorders.     

HIF-1α Dependent Upregulation of ZIP8, ZIP14, and TRPA1 Modify Intracellular Zn2+ Accumulation in Inflammatory Synoviocytes

Intracellular free zinc ([Zn²⁺]_i) is mobilized in neuronal and non-neuronal cells under physiological and/or pathophysiological conditions; therefore, [Zn²⁺]_i is a component of cellular signal transduction in biological systems. Although several transporters and ion channels that carry Zn²⁺ have been identified, proteins that are involved in Zn²⁺ supply into cells and their expression are poorly understood, particularly under inflammatory conditions. Here, we show that the expression of Zn²⁺ transporters ZIP8 and ZIP14 is increased via the activation of hypoxia-induced factor 1α (HIF-1α) in inflammation, leading to [Zn²⁺]_i accumulation, which intrinsically activates transient receptor potential ankyrin 1 (TRPA1) channel and elevates basal [Zn²⁺]_i. In human fibroblast-like synoviocytes (FLSs), treatment with inflammatory mediators, such as tumor necrosis factor-α (TNF-α) and interleukin-1α (IL-1α), evoked TRPA1-dependent intrinsic Ca²⁺ oscillations. Assays with fluorescent Zn²⁺ indicators revealed that the basal [Zn²⁺]_i concentration was significantly higher in TRPA1-expressing HEK cells and inflammatory FLSs. Moreover, TRPA1 activation induced an elevation of [Zn²⁺]_i level in the presence of 1 μM Zn²⁺ in inflammatory FLSs. Among the 17 out of 24 known Zn²⁺ transporters, FLSs that were treated with TNF-α and IL-1α exhibited a higher expression of ZIP8 and ZIP14. Their expression levels were augmented by transfection with an active component of nuclear factor-κB P65 and HIF-1α expression vectors, and they could be abolished by pretreatment with the HIF-1α inhibitor echinomycin (Echi). The functional expression of ZIP8 and ZIP14 in HEK cells significantly increased the basal [Zn²⁺]_i level. Taken together, Zn²⁺ carrier proteins, TRPA1, ZIP8, and ZIP14, induced under HIF-1α mediated inflammation can synergistically change [Zn²⁺]_i in inflammatory FLSs.     

The Anti-Atherosclerosis Effect of Anakinra,a Recombinant Human Interleukin-1 Receptor Antagonist,in Apolipoprotein E Knockout Mice

Interleukin (IL)-1β plays an important role in atherosclerosis pathogenesis. We aimed to investigate the effect of anakinra, a recombinant human IL-1 receptor antagonist, on the progression of atherosclerosis in apolipoprotein E knockout (ApoE^–/–) mice. ApoE^–/– mice (8-week male) were treated with saline (control), anakinra 10, 25, and 50 mg/kg, respectively (n = 10 in each group). Mice were fed a standard chow (4 weeks) followed by an atherogenic diet (35kcal% fat, 1.25% cholesterol, 12 weeks). Atheromatous plaques in ApoE^–/– mice and the expression of inflammatory genes and signaling pathways in human umbilical vein endothelial cells (HUVECs), rat aortic smooth muscle cells (RAOSMCs), and 3T3-L1 adipocytes were assessed. Anakinra reduced the plaque size of the aortic arch (30.6% and 25.2% at the 25 mg/kg and 50 mg/kg doses, both p < 0.05) and serum triglyceride in ApoE^–/– mice and suppressed inflammatory genes (IL-1β and IL-6) expressions in HUVECs and RAOSMCs (all p < 0.05). In RAOSMCs, anakinra reduced metalloproteinase-9 expression in a dose-dependent manner and inhibited cell migration. Anakinra-treated mice exhibited trends of lower CD68+ macrophage infiltration in visceral fat and monocyte chemoattractant protein-1 expression was reduced in 3T3-L1 adipocytes. Anakinra could be a useful component for complementary treatment with a standard regimen to reduce the residual cardiovascular risk.     

The Mucus Binding Factor Is Not Necessary for Lacticaseibacillus rhamnosus CRL1505 to Exert Its Immunomodulatory Activities in Local and Distal Mucosal Sites

Both viable and non-viable orally administered Lacticaseibacillus rhamnosus CRL1505 modulate immunity in local (intestine) and distal (respiratory) mucosal sites. So, intestinal adhesion and colonization are not necessary for this probiotic strain to exert its immunomodulatory effects. In this work, a mucus-binding factor knockout CRL1505 strain (ΔmbfCRL1505) was obtained and the lack of binding ability to both intestinal epithelial cells and mucin was demonstrated in vitro. In addition, two sets of in vivo experiments in 6-week-old Balb/c mice were performed to evaluate ΔmbfCRL1505 immunomodulatory activities. (A) Orally administered ΔmbfCRL1505 prior to intraperitoneal injection of the Toll-like receptor 3 (TLR3) agonist poly(I:C) significantly reduced intraepithelial lymphocytes (CD3⁺NK1.1⁺CD8αα⁺) and pro-inflammatory mediators (TNF-α, IL-6 and IL-15) in the intestinal mucosa. (B) Orally administered ΔmbfCRL1505 prior to nasal stimulation with poly(I:C) significantly decreased the levels of the biochemical markers of lung tissue damage. In addition, reduced recruitment of neutrophils and levels of pro-inflammatory mediators (TNF-α, IL-6 and IL-8) as well as increased IFN-β and IFN-γ in the respiratory mucosa were observed in ΔmbfCRL1505-treated mice when compared to untreated control mice. The immunological changes induced by the ΔmbfCRL1505 strain were not different from those observed for the wild-type CRL1505 strain. Although it is generally accepted that the expression of adhesion factors is necessary for immunobiotics to induce their beneficial effects, it was demonstrated here that the mbf protein is not required for L. rhamnosus CRL1505 to exert its immunomodulatory activities in local and distal mucosal sites. These results are a step forward towards understanding the mechanisms involved in the immunomodulatory capabilities of L. rhamnosus CRL1505.     

Anti-Inflammatory Activity of Oat Beta-Glucans in a Crohn’s Disease Model: Time- and Molar Mass-Dependent Effects

Background: The incidence of Crohn’s disease (CD) is increasing worldwide, and it has currently become a serious public health issue in society. The treatment of CD continues throughout a patient’s lifetime, and therefore, it is necessary to develop new, effective treatment methods, including dietotherapy. The present study aimed to determine the effects of consumption of oat beta-glucans with different molar mass on colon inflammation (colitis) in the early stages of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced CD in an animal model. Methods: Sprague–Dawley rats (control and TNBS-induced CD) were divided into three dietary groups and fed for 3 days (reflecting acute inflammation) or 7 days (reflecting remission) with a feed containing 1% low (βGl) or high (βGh) molar mass oat beta-glucan or a feed without this polysaccharide. The level of colon inflammatory markers and the expression of cytokines and their receptor genes were measured by ELISA and RT-PCR methods, respectively. Results: Acute inflammation or remission (3 or 7 days after TNBS administration, respectively) stages of experimentally induced CD were characterized by an increase in the level of inflammatory markers (IL-1, IL-6, IL-10, IL-12, TNF-α, CRP, MPO, COX, and PGE2) and the disruption of some cytokine signaling pathways as well as macro- and microscopic changes of colon tissue. The consumption of oat beta-glucans reduced the level of inflammatory markers and recovered the signaling pathways and histological changes, with stronger effects of βGl after 7 days of colitis. Conclusions: Dietary oat beta-glucans can reduce colitis at the molecular and organ level and accelerate CD remission.