Role of gut microbial-derived metabolites and other select agents on adipocyte browning.

AIMS: Metabolic disease is a multifaceted condition characterized by the disruption of numerous metabolic parameters within the host. Its prevalence has surged significantly in recent years and it has become a prominent non-communicable disease worldwide. The effect of gut microbiota on various beige fat induction is well studied, while the mechanisms behind the link remain unclear. Given that gut microbiota-derived metabolites (meta-metabolites) secreted in the gut serve as a key mode of communication with their host through direct circulation or indirect host physiology modification, understanding the effect of meta-metabolites on adipose tissue is essential. METHODOLOGY: In our previous in-vivo studies, we observed a correlation between gut microbiota and the formation of beige fat. In this study, we further aimed to validate this correlation by treating the adipocyte cell line (3T3-L1) with meta-metabolites collected from the cecum of mice exhibiting beige adipose tissue formation. Additionally, we treated the adipocyte cell line with known beige fat inducers (L-Rhamnose and Ginsenoside) to assess meta-metabolites' efficacy on beige fat formation. KEY FINDINGS: Upon treatment with the meta-metabolites from the antibiotic-treated mice, we observed a significant increase in lipid metabolism and beige-specific gene expression. Analyzing the metabolites in these cells revealed that a set of metabolites potentially govern adipocytes, contributing to a metabolically active state. These effects were at par or even better than those of cells treated with L-Rhamnose or Ginsenoside. SIGNIFICANCE: This research sheds light on the intricate interplay between microbial metabolites and adipose tissue, offering valuable clues for understanding and potentially manipulating these processes for therapeutic purposes.

Effects of constant darkness on behaviour and physiology of male and female mice.

A healthy state of life suggests not only a disease-free condition but also normal psychological functioning and behaviour. To maintain a healthy life, the duration of light exposure is a crucial factor. Perturbation of the standard light-dark cycle (LD: 12 h light-12 h dark in mice) may result in brain, behavioural and physiological abnormalities. The current study determined the effects of 3 and 5 weeks of constant darkness (DD: 00 h light-24 h dark) on the behaviour, hormones, prefrontal cortex (PFC) and metabolome of male and female C57BL/6 J mice. We also studied 3 weeks of restoration in LD following 5 weeks of DD exposure. The results revealed that 3 weeks of DD affected male mice more than females, and 5 weeks of DD had a comparable impact on behaviour, hormones and the PFC of male and female mice. After restoration in LD, the DD-induced changes reverted to time-matched LD conditions in male and female mice. Furthermore, metabolome analysis corroborated male and female mice's behavioural and molecular kinetics. The present study laid the foundation for understanding how DD affects behaviour and the PFC as a function of (a) time and (b) sex and described the roles of stress and sex hormones, cytokines, neurotrophins and metabolic pathways.

Vancomycin-Induced Changes in Host Immunity and Behavior: Comparative Genomic and Metagenomic Analysis in C57BL/6 and BALB/c Mice.

BACKGROUND: The consequence of treatment with antibiotics on the gut microbiota can be destructive. The antibiotics, however, can be utilized to understand the role of gut microbiota on the host physiology. AIM: Earlier, we reported the efficacy of vancomycin in gut microbiota perturbation. We continued to understand the effect of restoration kinetics of perturbed gut microbiota on the immunity and behavior of Th1 (C57BL/6)- and Th2 (BALB/c)-biased mice. METHODS: We studied restoration kinetics of the gut microbiota for two months following the withdrawal of vancomycin treatment in both mice strains. We analyzed cecal microbiome composition, different behavioral assays, and expression of select genes associated with stress and barrier function in gut and brain. RESULTS: Metagenomic analysis of gut microbiota revealed that the treatment with vancomycin caused a significant decrease in the relative abundance of Firmicutes and Bacteroidetes phyla with a time-dependent increase in Proteobacteria and Verrucomicrobia phyla. Maximum restoration (> 70%) of gut microbiota happened by the 15th day of withdrawal of vancomycin. BALB/c mice showed a more efficient restoration of gut microbiota compared to C57BL/6 mice. We established the correlation patterns of gut microbiota alteration and its effect on (a) the behavior of mice, (b) expression of key brain molecules, and (c) immunity-related genes. CONCLUSIONS: The results revealed that the gut microbiome profiling, behavior, and immune responses varied significantly between Th1- and Th2-biased mice. By withdrawing the treatment with vancomycin of major gut microbes, important physiological and behavioral changes of both mice strains returned to the normal (untreated control) level.

Vaginal microbiota of asymptomatic bacterial vaginosis and vulvovaginal candidiasis: Are they different from normal microbiota?

Vaginal microbiota contributes in maintaining and protecting the urogenital niche from infections and their sequelae. Despite extensive research, microbiome studies have often ignored asymptomatic bacterial vaginosis (BV) and vulvovaginal candidiasis (VVC). The present study aimed to explore the cultivable vaginal bacterial and mycological communities in women asymptomatic for BV and VVC using multiplex PCR and species-specific PCR. Vaginal swabs collected from 199 participants asymptomatic for urogenital infections, scored by Nugent criteria indicated 73.9% had normal microbiota, 11.6% intermediate and 14.5% BV. The most frequent Lactobacillus species in normal women were L. iners (69.4%), L. crispatus (24.5%), L. reuteri (20.4%). Women with BV colonized L. iners (62.1%); L. rhamnosus (41.4%); L. salivarius (13.7%) and L. reuteri (7.2%). Furthermore, L. crispatus was associated with normal microbiota, whereas L. iners was a frequent member of normal and dysbiotic microbiota. Lactobacillus abundance and species richness reduced in asymptomatic BV. Also L. crispatus, L. fermentum, L. acidophilus and L. delbruckii were absent in these women. L. iners significantly co-existed with other Lactobacillus species, indicating its failure in independently maintaining the healthy vaginal niche. Of 30.4% women detected with Candida, 72.1% constituted non-albicans Candida. Predominance of C. albicans increased from 18.4% in healthy to 60% in women with asymptomatic BV; whereas distribution of BV related bacteria did not vary across the groups. Heterogeneous population of lactobacilli in 80.8% of normal women calls attention towards cumulative effects of these species in safeguarding the vaginal microenvironment. Since the microbiota of asymptomatic BV was different from healthy, screening and management could be encouraged to avoid further complications of infections.

Comparative efficacy analysis of anti-microbial peptides, LL-37 and indolicidin upon conjugation with CNT, in human monocytes.

BACKGROUND: Antimicrobial peptides (AMPs) have the potential to serve as an alternative to antibiotic. AMPs usually exert bactericidal activity via direct killing of microbial pathogens. Reports have proposed that by harnessing innate immune activation, AMPs can regulate pathogen invasion and may control infection. It has been reported that AMPs could be utilized to activate the innate mucosal immune response in order to eliminate pathogenic infections. This way of controlling pathogen infection, by activating host immunity, confers the potential to the select AMPs to alleviate the problem of antibiotic resistance. Among various AMPs tested LL-37 and indolicidin, showed promise to be potential candidates for eliciting enhanced host innate immune responses. LL-37 and indolicidin had exhibited substantial innate immune activation in both human and murine macrophages. Dosage for each of the AMPs, however, was high with adverse side effects. RESULTS: In this study, we reported that upon conjugation with carbon nanotubes (CNT), each AMP remained biologically functional at a concentration that was 1000-fold less than the dosage required for free AMP to remain active in the cells. CONCLUSIONS: Current study also revealed that while indolicidin induced signalling events mediated through the TNFRSF1A pathway in THP1 cells, followed by activation of NFκB and c-JUN pathways, treatment of cells with LL-37 induced signalling events by activating IL1R, with subsequent activation of NFκB and NFAT2. Thp1 cells, primed with CNT conjugated LL-37 or indolicidin, are protected against Salmonella typhimurium infection at 16 h post challenge.

Antibiotic induced adipose tissue browning in C57BL/6 mice: An association with the metabolic profile and the gut microbiota.

AIMS: The use of antibiotics affects health. The gut microbial dysbiosis by antibiotics is thought to be an essential pathway to influence health. It is important to have optimized energy utilization, in which adipose tissues (AT) play crucial roles in maintaining health. Adipocytes regulate the balance between energy expenditure and storage. While it is known that white adipose tissue (WAT) stores energy and brown adipose tissue (BAT) produces energy by thermogenesis, the role of an intermediate AT plays an important role in balancing host internal energy. In the current study, we tried to understand how treating an antibiotic cocktail transforms WAT into BAT or, more precisely, into beige adipose tissue (BeAT). METHODS: Since antibiotic treatment perturbs the host microbiota, we wanted to understand the role of gut microbial dysbiosis in transforming WAT into BeAT in C57BL/6 mice. We further correlated the metabolic profile at the systemic level with this BeAT transformation and gut microbiota profile. KEY FINDINGS: In the present study, we have reported that the antibiotic cocktail treatment increases the Proteobacteria and Actinobacteria while reducing the Bacteroidetes phylum. We observed that prolonged antibiotic treatment could induce the formation of BeAT in the inguinal and perigonadal AT. The correlation analysis showed an association between the gut microbiota phyla, beige adipose tissue markers, and serum metabolites. SIGNIFICANCE: Our study revealed that the gut microbiota has a significant role in regulating the metabolic health of the host via microbiota-adipose axis communication.

Postnatal intestinal mucosa and gut microbial composition develop hand in hand: A mouse study.

BACKGROUND: During the early postnatal life, gut microbiota development experiences dynamic changes in their structural and functional composition. The postnatal period is the critical window to develop a host defense mechanism. The maturation of intestinal mucosal barrier integrity is one of the essential defense mechanisms to prevent the entry of pathogens. However, the co-development of intestinal microbial colonization, formation of barrier integrity, and intestinal epithelial cell layer is not entirely understood. METHODS: We studied the gut microbial composition and diversity using 16S rRNA marker gene-based sequencing in mice to understand postnatal age-dependent association kinetics between gut microbial and intestinal development. Next, we assessed the intestinal development by in vivo gut permeability assay, mRNA gene expression of different tight junction proteins and intestinal epithelial cell markers, goblet cells population, villus length, and cecal IgA quantification. RESULTS: Our results showed a significant shift in gut microbial structural and functional composition from postnatal day 14 onwards with early life Proteobacteria abundance. Relative abundance of Verrucomicrobia was maximum at postnatal day 14 and showed a gradual decrease over time. We also observed an age-dependent biphasic pattern in barrier integrity improvement and differentiation of intestinal epithelial cells (IECs). A significant improvement in barrier integrity between days 1 and 7 showed the host factor contribution, while that beyond day 14 revealed an association with changes in microbiota composition. Our temporal correlation analysis associated Bacteroidetes phylum with the mucosal barrier formation during postnatal development. CONCLUSIONS: The present study revealed the importance and interplay of host factors and the microbiome in gut development and intestinal mucosal homeostasis.

A comparative analysis of gut microbial dysbiosis by select antibiotics and DSS to understand the effects of perturbation on the host immunity and metabolism.

AIMS: Balanced gut microbial composition of the host plays a crucial role in maintaining harmony among various physiological processes to maintain physiological homeostasis. Immunity and metabolism are the two physiologies mainly controlled by the gut microbiota. Reports suggested that gut microbial composition and diversity alteration are the leading causes of the host's healthy homeostasis alteration or a diseased state. The extent of gut perturbation depends on the perturbing agents' strength, chemical nature, and mode of action. In the current report, we have studied the effects of different perturbing agents on gut microbial dysbiosis and its impact on host immunity and metabolism. MATERIALS AND METHODS: We studied the perturbation of gut microbial composition and diversity using next-generation sequencing and further investigated the changes in host immune and metabolic responses. KEY FINDINGS: Enrichment or abolition of a particular phylum or genus depended on the perturbing agents. In the current study, treatment with neomycin yielded an increase in the Bacteroidetes phylum. Vancomycin treatment caused a significant rise in Verrucomicrobia and Proteobacteria phyla. The treatment with AVNM and DSS caused a substantial increase in the Proteobacteria phylum. The gut microbial diversity was also lowest in AVNM treated group. The altered gut microbial composition ultimately altered the immune responses at localized and systemic levels of the host. Gut dysbiosis also changed the systemic level of SCFAs. SIGNIFICANCE: This study will help us understand how the enrichment of a particular phylum and genus maintains the host's immune responses and metabolism.

Postnatal 14D is the Key Window for Mice Intestinal Development- An Insight from Age-Dependent Antibiotic-Mediated Gut Microbial Dysbiosis Study.

The postnatal period is one of the critical windows for the structure-function development of the gastrointestinal tract and associated mucosal immunity. Along with other constituent members, recent studies suggest the contribution of gut microbiota in maintaining host health, immunity, and development. Although the gut microbiota's role in maintaining barrier integrity is known, its function in early life development still needs to be better understood. To understand the details of gut microbiota's effects on intestinal integrity, epithelium development, and immune profile, the route of antibiotic-mediated perturbation is taken. Mice on days 7(P7D), 14(P14D), 21(P21D) and 28(P28D) are sacrificed and 16S rRNA metagenomic analysis is performed. The barrier integrity, tight junction proteins (TJPs) expression, intestinal epithelial cell (IEC) markers, and inflammatory cytokines are analyzed. Results reveal a postnatal age-related impact of gut microbiota perturbation, with a gradual increase in the relative abundance of Proteobacteria and a reduction in Bacteroidetes and Firmicutes. Significant barrier integrity disruption, reduced TJPs and IECs marker expression, and increased systemic inflammation at P14D of AVNM-treated mice are found. Moreover, the microbiota transplantation shows recolonization of Verrucomicrobia, proving a causal role in barrier functions. The investigation reveals P14D as a critical period for neonatal intestinal development, regulated by specific microbiota composition.

Sex and Time: Important Variables for Understanding the Impact of Constant Darkness on Behavior, Brain, and Physiology.

The circadian clock can coordinate, regulate and predict physiology and behavior in response to the standard light-dark (LD: 12 h light and 12 h dark) cycle. If we alter the LD cycle by exposing mice to constant darkness (DD: 00 h light and 24 h dark), it can perturb behavior, the brain, and associated physiological parameters. The length of DD exposure and the sex of experimental animals are crucial variables that could alter the impact of DD on the brain, behavior, and physiology, which have not yet been explored. We exposed mice to DD for three and five weeks and studied their impact on (1) behavior, (2) hormones, (3) the prefrontal cortex, and (4) metabolites in male and female mice. We also studied the effect of three weeks of standard light-dark cycle restoration after five weeks of DD on the parameters mentioned above. We found that DD exposure was associated with anxiety-like behavior, increased corticosterone and pro-inflammatory cytokines (TNF-α, IL-6, and IL-1ß), downregulated neurotrophins (BDNF and NGF), and altered metabolites profile in a duration of DD exposure and sex-dependent manner. Females showed a more robust adaptation than males under DD exposure. Three weeks of restoration was adequate to establish homeostasis in both sexes. To the best of our knowledge, this study is the first of its kind to look at how DD exposure impacts physiology and behavior as a function of sex- and time. These findings would have translational value and may help in establishing sex-specific interventions for addressing DD-related psychological issues.