Vaginal Lactobacilli and Vaginal Dysbiosis-Associated Bacteria Differently Affect Cervical Epithelial and Immune Homeostasis and Anti-Viral Defenses

Persistent infection with High Risk-Human Papilloma Viruses (HR-HPVs) is a primary cause of cervical cancer worldwide. Vaginal-dysbiosis-associated bacteria were correlated with the persistence of HR-HPVs infection and with increased cancer risk. We obtained strains of the most represented bacterial species in vaginal microbiota and evaluated their effects on the survival of cervical epithelial cells and immune homeostasis. The contribution of each species to supporting the antiviral response was also studied. Epithelial cell viability was affected by culture supernatants of most vaginal-dysbiosis bacteria, whereas Lactobacillus gasseri or Lactobacillus jensenii resulted in the best stimulus to induce interferon-γ (IFN-γ) production by human mononuclear cells from peripheral blood (PBMCs). Although vaginal-dysbiosis-associated bacteria induced the IFN-γ production, they were also optimal stimuli to interleukin-17 (IL-17) production. A positive correlation between IL-17 and IFN-γ secretion was observed in cultures of PBMCs with all vaginal-dysbiosis-associated bacteria suggesting that the adaptive immune response induced by these strains is not dominated by T_H1 differentiation with reduced availability of IFN-γ, cytokine most effective in supporting virus clearance. Based on these results, we suggest that a vaginal microbiota dominated by lactobacilli, especially by L. gasseri or L. jensenii, may be able to assist immune cells with clearing HPV infection, bypasses the viral escape and restores immune homeostasis.     

BMP3 Alone and Together with TGF-β Promote the Differentiation of Human Mesenchymal Stem Cells into a Nucleus Pulposus-Like Phenotype

Human mesenchymal stem cells (MSCs) have the potential to differentiate into nucleus pulposus (NP)-like cells under specific stimulatory conditions. Thus far, the effects of bone morphogenetic protein 3 (BMP3) and the cocktail effects of BMP3 and transforming growth factor (TGF)-β on MSC proliferation and differentiation remain obscure. Therefore, this study was designed to clarify these unknowns. MSCs were cultured with various gradients of BMP3 and BMP3/TGF-β, and compared with cultures in basal and TGF-β media. Cell proliferation, glycosaminoglycan (GAG) content, gene expression, and signaling proteins were measured to assess the effects of BMP3 and BMP3/TGF-β on MSCs. Cell number and GAG content increased upon the addition of BMP3 in a dose-dependent manner. The expression of COL2A1, ACAN, SOX9, and KRT19 increased following induction with BMP3 and TGF-β, in contrast to that of COL1A1, ALP, OPN, and COMP. Smad3 phosphorylation was upregulated by BMP3 and TGF-β, but BMP3 did not affect the phosphorylation of extracellular-signal regulated kinase (ERK) 1/2 or c-Jun N-terminal kinase (JNK). Our results reveal that BMP3 enhances MSC proliferation and differentiation into NP-like cells, as indicated by increased cell numbers and specific gene expressions, and may also cooperate with TGF-β induced positive effects. These actions are likely related to the activation of TGF-β signaling pathway.     

Skullcapflavone II Suppresses TNF-α/IFN-γ-Induced TARC,MDC, and CTSS Production in HaCaT Cells

Skullcapflavone II (SFII), a flavonoid derived from Scutellaria baicalensis, has been reported to have anti-inflammatory properties. However, its therapeutic potential for skin inflammatory diseases and its mechanism are unknown. Therefore, this study aimed to investigate the effect of SFII on TNF-α/IFN-γ-induced atopic dermatitis (AD)-associated cytokines, such as thymus- and activation-regulated chemokine (TARC) and macrophage-derived chemokine (MDC). Co-stimulation with TNF-α/IFN-γ in HaCaT cells is a well-established model for induction of pro-inflammatory cytokines. We treated cells with SFII prior to TNF-α/IFN-γ-stimulation and confirmed that it significantly inhibited TARC and MDC expression at the mRNA and protein levels. Additionally, SFII also inhibited the expression of cathepsin S (CTSS), which is associated with itching in patients with AD. Using specific inhibitors, we demonstrated that STAT1, NF-κB, and p38 MAPK mediate TNF-α/IFN-γ-induced TARC and MDC, as well as CTSS expression. Finally, we confirmed that SFII significantly suppressed TNF-α/IFN-γ-induced phosphorylation of STAT1, NF-κB, and p38 MAPK. Taken together, our study indicates that SFII inhibits TNF-α/IFN-γ-induced TARC, MDC, and CTSS expression by regulating STAT1, NF-κB, and p38 MAPK signaling pathways.     

Interferon Beta Activity Is Modulated via Binding of Specific S100 Proteins

Interferon-β (IFN-β) is a pleiotropic cytokine used for therapy of multiple sclerosis, which is also effective in suppression of viral and bacterial infections and cancer. Recently, we reported a highly specific interaction between IFN-β and S100P lowering IFN-β cytotoxicity to cancer cells (Int J Biol Macromol. 2020; 143: 633–639). S100P is a member of large family of multifunctional Ca²⁺-binding proteins with cytokine-like activities. To probe selectivity of IFN-β—S100 interaction with respect to S100 proteins, we used surface plasmon resonance spectroscopy, chemical crosslinking, and crystal violet assay. Among the thirteen S100 proteins studied S100A1, S100A4, and S100A6 proteins exhibit strictly Ca²⁺-dependent binding to IFN-β with equilibrium dissociation constants, K_d, of 0.04–1.5 µM for their Ca²⁺-bound homodimeric forms. Calcium depletion abolishes the S100—IFN-β interactions. Monomerization of S100A1/A4/A6 decreases K_d values down to 0.11–1.0 nM. Interferon-α is unable of binding to the S100 proteins studied. S100A1/A4 proteins inhibit IFN-β-induced suppression of MCF-7 cells viability. The revealed direct influence of specific S100 proteins on IFN-β activity uncovers a novel regulatory role of particular S100 proteins, and opens up novel approaches to enhancement of therapeutic efficacy of IFN-β.     

Oral Administration of Bovine Milk from Cows Hyperimmunized with Intestinal Bacterin Stimulates Lamina Propria T Lymphocytes to Produce Th1-Biased Cytokines in Mice

The goal of this study was to examine the effects of oral administration of bovine milk from cows hyperimmunized with a proprietary bacterin (immune milk “Sustaina”) on mucosal immunity in the intestine of adult mice. C57BL/6 mice were orally given immune or control milk for two weeks, and then lymphocyte population and the cytokine production in lamina propria of colon in normal mice and mice induced colitis by dextran sulphate sodium (DSS) were detected. We found that the levels of IFN-γ and IL-10 increased, but the levels of IL-17A and IL-4, decreased in lamina propria of colon in immune milk-fed mice as compared with those in control milk-fed mice. Interestingly, oral administration of immune milk partially improved the acute colitis induced by DSS. The levels of TNF-α and IFN-γ increased, but IL-6, IL-17A and IL-4 decreased in lamina propria (LP) of colon in immune milk-fed mice with DSS-induced colitis. Our results suggest that immune milk may stimulate CD4⁺ T cells to polarize towards a Th1 type response, but contrarily suppress Th17 and Th2 cells responses in large intestinal LP of mice. The results indicate that this kind of immune milk has is able to promote the maintainance of intestinal homeostasis and enhance protection against infection, and could alleviate the symptoms of acute colitis in mice.     

Molecular Identification of the G-Protein Genes and Their Expression Profiles in Response to Nitrogen Deprivation in Brassica napus

Heterotrimeric guanine nucleotide binding protein (G-protein) consisting of Gα, Gβ, and Gγ subunits is one of the key signal transducers in plants. Recent studies indicated that G-protein has been proposed as an important mediator of nitrogen responses in rice, wheat, and Arabidopsis. However, little is known about these G-proteins in Brassica napus (B. napus), except for three identified G-proteins, BnGA1, BnGB1, and BnGG2. Therefore, the aim of the present study is to characterize the members of the G-protein gene family in allotetraploid B. napus and to analyze their expression profiles in response to nitrogen deprivation. In total, 21 G-protein family members were identified in B. napus, encoding two Gα, six Gβ, and 13 Gγ. Sequence and phylogenetic analyses showed that although genome-wide triploid events increased the number of genes encoding Gα, Gβ, and Gγ subunits, the gene structure and protein properties of the genes encoding each G-protein subunit were extremely conserved. Collinearity analysis showed that most G-protein genes in B. napus had syntenic relationships with G-protein members of Arabidopsis, Brassica rape (B. rapa), and Brassica oleracea (B. oleracea). Expression profile analysis indicated that Gα and C-type Gγ genes (except BnGG10 and BnGG12 were highly expressed in flower and ovule) were barely expressed in most organs, whereas most Gβ and A-type Gγ genes tended to be highly expressed in most organs. G-protein genes also showed various expression patterns in response to nitrogen-deficient conditions. Under nitrogen deficiency, Gα and five C-type Gγ genes were upregulated initially in roots, while in leaves, Gα was downregulated initially and five C-type Gγ genes were highly expressed in different times. These results provide a complex genetic dissection of G-protein genes in B. napus, and insight into the biological functions of G-protein genes in response to nitrogen deficiency.     

Mycophenolate Antagonizes IFN-γ-Induced Catagen-Like Changes via β-Catenin Activation in Human Dermal Papilla Cells and Hair Follicles

Recently, various immunosuppressant drugs have been shown to induce hair growth in normal hair as well as in alopecia areata and androgenic alopecia; however, the responsible mechanism has not yet been fully elucidated. In this study, we investigate the influence of mycophenolate (MPA), an immunosuppressant, on the proliferation of human dermal papilla cells (hDPCs) and on the growth of human hair follicles following catagen induction with interferon (IFN)-γ. IFN-γ was found to reduce β-catenin, an activator of hair follicle growth, and activate glycogen synthase kinase (GSK)-3β, and enhance expression of the Wnt inhibitor DKK-1 and catagen inducer transforming growth factor (TGF)-β2. IFN-γ inhibited expression of ALP and other dermal papillar cells (DPCs) markers such as Axin2, IGF-1, and FGF 7 and 10. MPA increased β-catenin in IFN-γ-treated hDPCs leading to its nuclear accumulation via inhibition of GSK3β and reduction of DKK-1. Furthermore, MPA significantly increased expression of ALP and other DPC marker genes but inhibited expression of TGF-β2. Therefore, we demonstrate for the first time that IFN-γ induces catagen-like changes in hDPCs and in hair follicles via inhibition of Wnt/β-catenin signaling, and that MPA stabilizes β-catenin by inhibiting GSK3β leading to increased β-catenin target gene and DP signature gene expression, which may, in part, counteract IFN-γ-induced catagen in hDPCs.     

Modulation of Gut Microbiota Combined with Upregulation of Intestinal Tight Junction Explains Anti-Inflammatory Effect of Corylin on Colitis-Associated Cancer in Mice

Inflammatory bowel disease (IBD) involves chronic inflammation, loss of epithelial integrity, and gastrointestinal microbiota dysbiosis, resulting in the development of a colon cancer known as colitis-associated colorectal cancer (CAC). In this study, we evaluated the effects of corylin in a mouse model of dextran sodium sulfate (DSS)-induced colitis. The results showed corylin could improved the survival rate and colon length, maintained body weight, and ameliorated the inflammatory response in the colon. Then, we further identified the possible antitumor effects after 30-day treatment of corylin on an azoxymethane (AOM)/DSS-induced CAC mouse model. Biomarkers associated with inflammation, the colon tissue barrier, macrophage polarization (CD11c, CCR7, CD163, and CD206), and microbiota dysbiosis were monitored in the AOM/DSS group versus corylin groups. Corylin downregulated pro-inflammatory cytokines (TNF-α, IFN-γ, IL-1β, and IL-6) mRNA expression and inflammatory signaling-associated markers (TLR4, MyD88, AP-1, CD11b, and F4/80). In addition, a colon barrier experiment revealed that epithelial cell proliferation of the mucus layer (Lgr5, Cyclin D1, and Olfm4) was downregulated and tight junction proteins (claudin-1 and ZO-1) were upregulated. Furthermore, the Firmicutes/Bacteroidetes ratio changed with corylin intervention, and the microbial diversity and community richness of the AOM/DSS mice were improved by corylin. The comparative analysis of gut microbiota revealed that Bacteroidetes, Patescibacteria, Candidatus Saccharimonas, Erysipelatoclostridium, and Enterorhabdus were significantly increased but Firmicutes, Turicibacter, Romboutsia, and Blautia decreased after corylin treatment. Altogether, corylin administration showed cancer-ameliorating effects by reducing the risk of colitis-associated colon cancer via regulation of inflammation, carcinogenesis, and compositional change of gut microbiota. Therefore, corylin could be a novel, potential health-protective, natural agent against CAC.     

Modulation of PTPN2/22 Function by Spermidine in CRISPR-Cas9-Edited T-Cells Associated with Crohn’s Disease and Rheumatoid Arthritis

Crohn’s Disease (CD) and Rheumatoid Arthritis (RA) share some single nucleotide polymorphisms (SNPs) in protein tyrosine phosphatase non-receptor types 2 and 22 (PTPN2/22). Recently, we reported that clinical samples from CD and RA patients associated with PTPN2:rs478582 or PTPN22:rs2476601 genotypes were linked to overactive immune response and exacerbation of inflammation. Here, we investigated in vitro the effects of these SNPs in Jurkat T-cells using CRISPR-Cas9. All cells were evaluated for PTPN22/22 loss of function and effects on cell response. We measured gene expression via RT-qPCR and cytokines by ELISA. We also measured cell proliferation using a BrdU labeling proliferation ELISA, and T-cell activation using CD-25 fluorescent immunostaining. In PTPN2 SNP-edited cells, PTPN2 expression decreased by 3.2-fold, and proliferation increased by 10.2-fold compared to control. Likewise, expression of PTPN22 decreased by 2.4-fold and proliferation increased by 8.4-fold in PTPN22 SNP-edited cells. IFN-γ and TNF-α secretions increased in both edited cell lines. CD25 expression (cell activation) was 80.32% in PTPN2 SNP-edited cells and 85.82% in PTPN22 SNP-edited cells compared to 70.48% in unedited Jurkat T-cells. Treatment of PTPN2 and PTPN22-edited cells with a maximum 20 μM spermidine restored PTPN2/22 expression and cell response including cell proliferation, activation, and cytokines secretion. Most importantly, the effect of spermidine on edited cells restored normal expression and secretion of IFN-γ and TNF-α. The data clearly demonstrated that edited SNPs in PTPN2 or PTPN22 were associated with reduced gene expression, which resulted in an increase in cell proliferation and activation and overactive immune response. The data validated our earlier observations in CD and RA clinical samples. Surprisingly, spermidine restored PTPN2/22 expression in edited Jurkat T-cells and the consequent beneficial effect on cell response and inflammation. The study supports the use of polyamines dietary supplements for management of CD and in RA patients.     

Ethyl Gallate Dual-Targeting PTPN6 and PPARγ Shows Anti-Diabetic and Anti-Obese Effects

The emergence of the high correlation between type 2 diabetes and obesity with complicated conditions has led to the coinage of the term “diabesity”. AMP-activated protein kinase (AMPK) activators and peroxisome proliferator-activated receptor (PPARγ) antagonists have shown therapeutic activity for diabesity, respectively. Hence, the discovery of compounds that activate AMPK as well as antagonize PPARγ may lead to the discovery of novel therapeutic agents for diabesity. In this study, the knockdown of PTPN6 activated AMPK and suppressed adipogenesis in 3T3-L1 cells. By screening a library of 1033 natural products against PTPN6, we found ethyl gallate to be the most selective inhibitor of PTPN6 (K_i = 3.4 μM). Subsequent assay identified ethyl gallate as the best PPARγ antagonist (IC₅₀ = 5.4 μM) among the hit compounds inhibiting PTPN6. Ethyl gallate upregulated glucose uptake and downregulated adipogenesis in 3T3-L1 cells as anticipated. These results strongly suggest that ethyl gallate, which targets both PTPN6 and PPARγ, is a potent therapeutic candidate to combat diabesity.     

Characterization of the Transient Deficiency of PKC Isozyme Levels in Immature Cord Blood T Cells and Its Connection to Anti-Allergic Cytokine Profiles of the Matured Cells

Cord blood T cells (CBTC) from a proportion of newborns express low/deficient levels of some protein kinase C (PKC) isozymes, with low levels of PKCζ correlating with increased risk of developing allergy and associated decrease in interferon-gamma (IFN-γ) producing T cells. Interestingly, these lower levels of PKCζ were increased/normalized by supplementing women during pregnancy with n-3 polyunsaturated fatty acids. However, at present, we have little understanding of the transient nature of the deficiency in the neonate and how PKCζ relates to other PKC isozymes and whether their levels influence maturation into IFN-γ producing T cells. There is also no information on PKCζ isozyme levels in the T cell subpopulations, CD4⁺ and CD8⁺ cells. These issues were addressed in the present study using a classical culture model of neonatal T cell maturation, initiated with phytohaemagglutinin (PHA) and recombinant human interleukin-2 (rhIL-2). Of the isozymes evaluated, PKCζ, β2, δ, μ, ε, θ and λ/ι were low in CBTCs. The PKC isozyme deficiencies were also found in the CD4⁺ and CD8⁺ T cell subset levels of the PKC isozymes correlated between the two subpopulations. Examination of changes in the PKC isozymes in these deficient cells following addition of maturation signals showed a significant increase in expression within the first few hours for PKCζ, β2 and μ, and 1–2 days for PKCδ, ε, θ and λ/ι. Only CBTC PKCζ isozyme levels correlated with cytokine production, with a positive correlation with IFN-γ, interleukin (IL)-2 and tumour necrosis factor-alpha (TNF), and a negative association with IL-9 and IL-10. The findings reinforce the specificity in using CBTC PKCζ levels as a biomarker for risk of allergy development and identify a period in which this can be potentially ‘corrected’ after birth.     

Ex Vivo Human Colon Tissue Exposure to Pristine Graphene Activates Genes Involved in the Binding,Adhesion and Proliferation of Epithelial Cells

Toxicology studies on pristine graphene are limited and lack significant correlations with actual human response. The goal of the current study was to determine the response of total colonic human tissue to pristine graphene exposure. Biopsy punches of colon tissues from healthy human were used to assess the biological response after ex vivo exposure to graphene at three different concentrations (1, 10, and 100 µg/mL). mRNA expression of specific genes or intestinal cytokine abundance was assessed using real-time PCR or multiplex immunoassays, respectively. Pristine graphene-activated genes that are related to binding and adhesion (GTPase and KRAS) within 2 h of exposure. Furthermore, the PCNA (proliferating cell nuclear antigen) gene was upregulated after exposure to graphene at all concentrations. Ingenuity pathway analysis revealed that STAT3 and VEGF signaling pathways (known to be involved in cell proliferation and growth) were upregulated. Graphene exposure (10 µg/mL) for 24 h significantly increased levels of pro-inflammatory cytokines IFNγ, IL-8, IL-17, IL-6, IL-9, MIP-1α, and Eotaxin. Collectively, these results indicated that graphene may activate the STAT3–IL23–IL17 response axis. The findings in this study provide information on toxicity evaluation using a human-relevant ex vivo colon model and serve as a basis for further exploration of its bio-applications.     

Identification of a Tumor Cell Associated Type I IFN Resistance Gene Expression Signature of Human Melanoma,the Components of Which Have a Predictive Potential for Immunotherapy

We developed a human melanoma model using the HT168-M1 cell line to induce IFN-α2 resistance in vitro (HT168-M1res), which was proven to be maintained in vivo in SCID mice. Comparing the mRNA profile of in vitro cultured HT168-M1res cells to its sensitive counterpart, we found 79 differentially expressed genes (DEGs). We found that only a 13-gene core of the DEGs was stable in vitro and only a 4-gene core was stable in vivo. Using an in silico cohort of IFN-treated melanoma tissues, we validated a differentially expressed 9-gene core of the DEGs. Furthermore, using an in silico cohort of immune checkpoint inhibitor (ICI)-treated melanoma tissues, we tested the predictive power of the DEGs for the response rate. Analysis of the top four upregulated and top four downregulated genes of the DEGs identified WFDC1, EFNA3, DDX10, and PTBP1 as predictive genes, and analysis of the “stable” genes of DEGs for predictive potential of ICI response revealed another 13 genes, out of which CDCA4, SOX4, DEK, and HSPA1B were identified as IFN-regulated genes. Interestingly, the IFN treatment associated genes and the ICI-therapy predictive genes overlapped by three genes: WFDC1, BCAN, and MT2A, suggesting a connection between the two biological processes.     

Design,Synthesis and Evaluation of Novel Derivatives of Curcuminoids with Cytotoxicity

Curcumin and curcuminoids have been discussed frequently due to their promising functional groups (such as scaffolds of α,β-unsaturated β-diketone, α,β-unsaturated ketone and β′-hydroxy-α,β-unsaturated ketone connected with aromatic rings on both sides) that play an important role in various bioactivities, including antioxidant, anti-inflammatory, anti-proliferation and anticancer activity. A series of novel curcuminoid derivatives (a total of 55 new compounds) and three reference compounds were synthesized with good yields using three-step organic synthesis. The anti-proliferative activities of curcumin derivatives were examined for six human cancer cell lines: HeLaS3, KBvin, MCF-7, HepG2, NCI-H460 and NCI-H460/MX20. Compared to the IC₅₀ values of all the synthesized derivatives, most α,β-unsaturated ketones displayed potent anti-proliferative effects against all six human cancer cell lines, whereas β′-hydroxy-α,β-unsaturated ketones and α,β-unsaturated β-diketones presented moderate anti-proliferative effects. Two potent curcuminoid derivatives were found among all the novel derivatives and reference compounds: (E)-5-hydroxy-7-phenyl-1-(3,4,5-trimethoxyphenyl)hept-1-en-3-one (compound 3) and (1E,4E)-1,7-bis(3,4,5-trimethoxyphenyl)hepta-1,4-dien-3-one (compound MD12a). These were selected for further analysis after the evaluation of their anti-proliferative effects against all human cancer cell lines. The results of apoptosis assays revealed that the number of dead cells was increased in early apoptosis and late apoptosis, while cell proliferation was also decreased after applying various concentrations of (E)-5-hydroxy-7-phenyl-1-(3,4,5-trimethoxyphenyl)hept-1-en-3-one (compound 3) and (1E,4E)-1,7-bis(3,4,5-trimethoxyphenyl)hepta-1,4-dien-3-one (compound MD12a) to MCF-7 and HpeG2 cancer cells. Analysis of the gene expression arrays showed that three genes (GADD45B, SESN2 and BBC3) were correlated with the p53 pathway. From the quantitative PCR analysis, it was seen that (1E,4E)-1,7-bis(3,4,5-trimethoxyphenyl)hepta-1,4-dien-3-one (compound MD12a) effectively induced the up-regulated expression of GADD45B, leading to the suppression of MCF-7 cancer cell formation and cell death. Molecular docking analysis was used to predict and sketch the interactions of the GADD45B-α,β-unsaturated ketone complex for help in drug design.