The Regulatory Roles of Ezh2 in Response to Lipopolysaccharide (LPS) in Macrophages and Mice with Conditional Ezh2 Deletion with LysM-Cre System.

The responses of macrophages to lipopolysaccharide (LPS) might determine the direction of clinical manifestations of sepsis, which is the immune response against severe infection. Meanwhile, the enhancer of zeste homologue 2 (Ezh2), a histone lysine methyltransferase of epigenetic regulation, might interfere with LPS response. Transcriptomic analysis on LPS-activated wild-type macrophages demonstrated an alteration of several epigenetic enzymes. Although the Ezh2-silencing macrophages (RAW264.7), using small interfering RNA (siRNA), indicated a non-different response to the control cells after a single LPS stimulation, the Ezh2-reducing cells demonstrated a less severe LPS tolerance, after two LPS stimulations, as determined by the higher supernatant TNF-α. With a single LPS stimulation, Ezh2 null (Ezh2flox/flox; LysM-Crecre/-) macrophages demonstrated lower supernatant TNF-α than Ezh2 control (Ezh2fl/fl; LysM-Cre-/-), perhaps due to an upregulation of Socs3, which is a suppressor of cytokine signaling 3, due to the loss of the Ezh2 gene. In LPS tolerance, Ezh2 null macrophages indicated higher supernatant TNF-α and IL-6 than the control, supporting an impact of the loss of the Ezh2 inhibitory gene. In parallel, Ezh2 null mice demonstrated lower serum TNF-α and IL-6 than the control mice after an LPS injection, indicating a less severe LPS-induced hyper-inflammation in Ezh2 null mice. On the other hand, there were similar serum cytokines after LPS tolerance and the non-reduction of serum cytokines after the second dose of LPS, indicating less severe LPS tolerance in Ezh2 null mice compared with control mice. In conclusion, an absence of Ezh2 in macrophages resulted in less severe LPS-induced inflammation, as indicated by low serum cytokines, with less severe LPS tolerance, as demonstrated by higher cytokine production, partly through the upregulated Socs3.

Analysis of cutaneous bacterial microbiota of Thai patients with seborrheic dermatitis.

Seborrheic dermatitis (SD) is a chronic inflammatory skin condition that occurs in body areas that contain profuse sebaceous glands. Skin microbiota are diverse across ethnic groups and its dysbiosis has been implicated in the pathogenesis of SD. Here, we reported the contribution of cutaneous bacterial microbiota to SD in the Thai population. Healthy individuals and patients with scalp SD were recruited into the study. Normal skin, scalp skin lesion (SL) and non-lesion sites (SNL) samples were collected using a tape stripping method and next-generation sequencing of 16S rRNA for microbiome analysis. Although bacterial diversity in all sample groups was not statistically different, a population of bacteria commonly found on skin of scalp showed signs of dysbiosis. Apart from the reduction of Corynebacterium spp., SD-specific microbiota was dominated by Firmicutes at taxa level and Pseudomonas spp., Staphylococcus spp. and Micrococcus spp. at genus level. The dysbiosis of the skin microbiota in SD was specifically described as an alteration of bacteria populations commonly found on scalp skin, implying that managing and controlling the cutaneous bacterial microbiome can alleviate and prevent SD and pave the way for the development of new SD treatments.

The immediate patch test reaction to fragrance in patients with allergic contact dermatitis to fragrance: A prospective study.

BACKGROUND: Fragrance is one of the common causes of immediate contact reaction. Knowing the prevalence of a reaction in a given population enables prioritization of allergy screening. OBJECTIVE: The purpose of this study was to determine the prevalence of an immediate patch test reaction to fragrance in patients with fragrance allergic contact dermatitis. METHODS: This prospective study enrolled 291 patients who were given standard patch tests for allergic contact dermatitis. Those with positive reactions were then asked to undergo additional patch tests to assess both immediate and delayed reactions to 28 different fragrance substances. RESULTS: Cinnamic aldehyde and cinnamic alcohol were the most frequently encountered substances in positive immediate reactions and standard (delayed) patch test reactions. Immediate patch reactions to benzyl alcohol, sorbic acid, and coumarin were more frequently observed than standard patch test reactions. LIMITATIONS: Because of the small sample size of patients who agreed to continue further patch testing evaluation, a statistical association between patient characteristics and fragrance-positive patch test reactions was difficult to establish. CONCLUSIONS: In this population, cinnamic aldehyde and cinnamic alcohol were the most common fragrance allergens causing both immediate and delayed reactions, whereas reactions to benzyl alcohol, sorbic acid, and coumarin were frequently observed in immediate patch tests.

Blood Bacteria-Free DNA in Septic Mice Enhances LPS-Induced Inflammation in Mice through Macrophage Response.

Although bacteria-free DNA in blood during systemic infection is mainly derived from bacterial death, translocation of the DNA from the gut into the blood circulation (gut translocation) is also possible. Hence, several mouse models with experiments on macrophages were conducted to explore the sources, influences, and impacts of bacteria-free DNA in sepsis. First, bacteria-free DNA and bacteriome in blood were demonstrated in cecal ligation and puncture (CLP) sepsis mice. Second, administration of bacterial lysate (a source of bacterial DNA) in dextran sulfate solution (DSS)-induced mucositis mice elevated blood bacteria-free DNA without bacteremia supported gut translocation of free DNA. The absence of blood bacteria-free DNA in DSS mice without bacterial lysate implies an impact of the abundance of bacterial DNA in intestinal contents on the translocation of free DNA. Third, higher serum cytokines in mice after injection of combined bacterial DNA with lipopolysaccharide (LPS), when compared to LPS injection alone, supported an influence of blood bacteria-free DNA on systemic inflammation. The synergistic effects of free DNA and LPS on macrophage pro-inflammatory responses, as indicated by supernatant cytokines (TNF-α, IL-6, and IL-10), pro-inflammatory genes (NFκB, iNOS, and IL-1ß), and profound energy alteration (enhanced glycolysis with reduced mitochondrial functions), which was neutralized by TLR-9 inhibition (chloroquine), were demonstrated. In conclusion, the presence of bacteria-free DNA in sepsis mice is partly due to gut translocation of bacteria-free DNA into the systemic circulation, which would enhance sepsis severity. Inhibition of the responses against bacterial DNA by TLR-9 inhibition could attenuate LPS-DNA synergy in macrophages and might help improve sepsis hyper-inflammation in some situations.

Coculturing liver cancer cells and monocytes in spheroids conditions monocytes to adopt tumor-associated macrophage phenotypes that favor tumor growth via cholesterol metabolism.

Tumor-infiltrating immune cells and their crosstalk with cancer cells in the tumor microenvironment (TME) play a crucial role in shaping tumor progression and response to therapy. We utilized 3-dimensional liver cancer spheroids incorporating human primary monocytes to investigate the crosstalk between tumor-associated macrophages (TAMs) and Hepatocellular carcinoma (HCC) cells, HepG2 and PLC/PRF/5. Using multiplexed gene expression panels, the critical pathways involved in shaping primary human monocytes to adopt TAMs phenotypes were identified. The specific inhibitor for an identified pathway was used to explore its involvement in polarization of TAMs. In the cocultured spheroids comprising the human HCC cell lines, the infiltrating monocytes resembled protumor M2-like macrophage phenotypes. Gene expression panels of the infiltrating monocytes demonstrated that the upregulated genes were enriched in the cholesterol metabolism pathway. Cholesterol metabolism-related genes were upregulated together with the nuclear receptors, PPARG and LXR. When lysosomal acid lipase (LAL), the key enzyme necessary for the hydrolysis of lipoprotein, was inhibited, infiltrating monocytes in 3-dimensional spheroid coculture showed significantly decreased M2 marker and lipid uptake receptor expression as well as increased cellular lipid content, which indicated that cholesterol metabolism was important for conditioning the TAMs. Moreover, LAL inhibition reduced the spheroid growth and invasiveness of HCC cell lines. Small interfering RNA-mediated LAL silencing in monocytes yielded similar results upon spheroid coculture. These data indicated that liver cancer cells and infiltrating monocytes participate in crosstalk via cholesterol metabolism to condition monocytes toward TAMs, which favors tumor growth and survival, thereby promoting liver cancer progression.

Live-cell imaging Unveils stimulus-specific dynamics of Nrf2 activation in UV-exposed melanoma cells: Implications for antioxidant compound screening.

The transcription factor Nuclear factor e2-related factor 2 (Nrf2) is pivotal in orchestrating cellular antioxidant defense mechanisms, particularly in skin cells exposed to ultraviolet (UV) radiation and electrophilic phytochemicals. To comprehensively investigate Nrf2's role in maintaining cellular redox equilibrium following UV-induced stress, we engineered a novel Nrf2 fusion-based reporter system for real-time, live-cell quantification of Nrf2 activity in human melanoma cells. Utilizing live quantitative imaging, we dissected the kinetic profiles of Nrf2 activation in response to an array of stimuli, including UVA and UVB radiation, as well as a broad spectrum of phytochemicals including ferulic acid, gallic acid, hispidulin, p-coumaric acid, quercetin, resveratrol, tannic acid, and vanillic acid as well as well-known Nrf2 inducers, tert-butylhydroquinone (tBHQ) and sulforaphane (SFN). Intriguingly, we observed distinct dynamical patterns of Nrf2 activity contingent on the specific stimuli applied. Sustained activation of Nrf2 was empirically correlated with the increased antioxidant response element (ARE) activity. Our findings demonstrate the nuanced impact of different phenolic compounds on Nrf2 activity and the utility of our Nrf2-CTΔ16-YFP reporter in characterizing the dynamics of Nrf2 translocation in response to diverse stimuli. In summary, our innovative reporter system not only revealed compounds capable of modulating UVA-induced Nrf2 activity but also showcased its utility as a robust tool for future antioxidant compound screening efforts.

O6-methylguanine DNA methyltransferase regulates ß-glucan-induced trained immunity of macrophages via farnesoid X receptor and AMPK.

Trained immunity is the heightened state of innate immune memory that enhances immune response resulting in nonspecific protection. Epigenetic changes and metabolic reprogramming are critical steps that regulate trained immunity. In this study, we reported the involvement of O6-methylguanine DNA methyltransferase (MGMT), a DNA repair enzyme of lesion induced by alkylating agents, in regulation the trained immunity induced by ß-glucan (BG). Pharmacological inhibition or silencing of MGMT expression altered LPS stimulated pro-inflammatory cytokine productions in BG-trained bone marrow derived macrophages (BMMs). Targeted deletion of Mgmt in BMMs resulted in reduction of the trained responses both in vitro and in vivo models. The transcriptomic analysis revealed that the dampening trained immunity in MGMT KO BMMs is partially mediated by ATM/FXR/AMPK axis affecting the MAPK/mTOR/HIF1α pathways and the reduction in glycolysis function. Taken together, a failure to resolve a DNA damage may have consequences for innate immune memory.

Sentiment and Topic Modeling Analysis on Twitter Reveals Concerns over Cannabis-Containing Food after Cannabis Legalization in Thailand.

OBJECTIVES: Twitter has been used to express a diverse range of public opinions about cannabis legalization in Thailand. The purpose of this study was to observe changes in sentiments after cannabis legalization and to investigate health-related topics discussed on Twitter. METHODS: Tweets in Thai and English related to cannabis were scraped from Twitter between May 1 and June 13, 2022, during cannabis legalization in Thailand. Sentiment and topic-modeling analyses were used to compare the content of tweets before and after legalization. Health-related topics were manually grouped into categories by their content and rated according to the number of corresponding tweets. RESULTS: We collected 21,242 and 6,493 tweets, respectively, for Thai and English search terms. A sharp increase in the number of tweets related to cannabis legalization was detected at the time of its public announcement. Sentiment analysis in the Thai search group showed a significant change (p < 0.0001) in sentiment distribution after legalization, with increased negative and decreased positive sentiments. A significant change was not found in the English search group (p = 0.4437). Regarding cannabis-containing food as a leading issue, topic-modeling analysis revealed public concerns after legalization in the Thai search group, but not the English one. Topics related to cannabis tourism surfaced only in the English search group. CONCLUSIONS: Since cannabis legalization, the primary health-related concern has been cannabis-containing food. Education and clear regulations on cannabis use are required to strengthen oversight of cannabis in the Thai population, as well as among medical tourists.

Impaired functions of human monocyte-derived dendritic cells and induction of regulatory T cells by pathogenic Leptospira.

Leptospirosis is a global zoonosis caused by pathogenic Leptospira. The disease outcome is influenced by the interplay between innate and adaptive immune responses. Dendritic cells (DCs) play a crucial role in shaping the adaptive immune response. A recent study revealed that pathogenic Leptospira limited the activation of human monocyte-derived dendritic cells (MoDCs) compared to non-pathogenic Leptospira, but their impact on T-cell responses has not been investigated. Our study is the first to explore how viable pathogenic and non-pathogenic Leptospira affect the interaction between human MoDCs and T cells. We found that MoDCs infected with pathogenic leptospires (L. interrogans serovar Pomona and a clinical isolate, MoDCs-P) exhibited lower levels of CD80 and CD83 expression, suggesting partially impaired MoDC maturation, induced regulatory T cells (Tregs) while failing to induce CD4+ T cell proliferation, compared to MoDCs infected with non-pathogenic leptospires (L. biflexa serovar Patoc and L. meyeri serovar Ranarum, MoDCs-NP). In contrast, non-pathogenic leptospires enhanced MoDC maturation and induced higher T cell proliferation including IFN-γ-producing CD4+ T cells, indicative of a Th1-type response. Furthermore, pathogenic leptospires induced higher MoDC apoptosis through a cysteine aspartic acid-specific protease-3 (caspase-3)-dependent pathway and upregulated expression of the prostaglandin-endoperoxide synthase 2 (PTGS2) gene. Notably, prostaglandin E2 (PGE2), a product of the PTGS2 pathway, was found at higher levels in the sera of patients with acute leptospirosis and in the supernatant of MoDCs-P, possibly contributing to Treg induction, compared to those of healthy donors and MoDCs-NP, respectively. In conclusion, this study reveals a novel immunosuppressive strategy employed by pathogenic Leptospira to evade host immunity by partially impairing MoDC maturation and inducing Tregs. These findings deepen our understanding of leptospirosis pathogenesis in humans and may provide a novel strategy to modulate DCs for the prevention and treatment of the disease.

Cyclic GMP-AMP Synthase (cGAS) Deletion Reduces Severity in Bilateral Nephrectomy Mice through Changes in Neutrophil Extracellular Traps and Mitochondrial Respiration.

Uremia-induced systemic inflammation is partly caused by the dissemination of microbial molecules such as lipopolysaccharide and bacterial double-stranded DNA from leaked gut damaged by immune cells in response to the microbial molecules. Cyclic GMP-AMP synthase (cGAS) can recognize fragmented DNA and induce cGAMP synthesis for the activation of the stimulator of interferon genes (STING) pathway. To study the effect of cGAS in uremia-induced systemic inflammation, we performed bilateral nephrectomy (BNx) in wild-type and cGAS knock-out mice and found that the gut leakage and blood uremia from both groups were similar. However, serum cytokines (TNF-α and IL-6) and neutrophil extracellular traps (NETs) decreased significantly in cGAS-/- neutrophils after stimulation with LPS or bacterial cell-free DNA. Transcriptomic analysis of LPS-stimulated cGAS-/- neutrophils also confirmed the down-regulation of neutrophil effector functions. The extracellular flux analysis showed that cGAS-/- neutrophils exhibited a higher respiratory rate than wild-type neutrophils despite having similar mitochondrial abundance and function. Our results suggest that cGAS may control effector functions and the mitochondrial respiration of neutrophils in response to LPS or bacterial DNA.