Background Acute cutaneous inflammation causes microbiome alterations as well as ultrastructural changes in epidermis stratification. However, the interactions between keratinocyte proliferation and differentiation status and the skin microbiome have not been fully explored. Objectives Hypothesizing that the skin microbiome contributes to regulation of keratinocyte differentiation and can modify antimicrobial responses, we examined the effect of exposure to commensal (Staphylococcus epidermidis, SE) or pathogenic (Staphylococcus aureus, SA) challenge on epidermal models. Methods Explant biopsies were taken to investigate species-specific antimicrobial effects of host factors. Further investigations were performed in reconstituted epidermal models by bulk transcriptomic analysis alongside secreted protein profiling. Single-cell RNA sequencing analysis was performed to explore the keratinocyte populations responsible for SA inflammation. A dataset of 6391 keratinocytes from control (2044 cells), SE challenge (2028 cells) and SA challenge (2319 cells) was generated from reconstituted epidermal models. Results Bacterial lawns of SA, not SE, were inhibited by human skin explant samples, and microarray analysis of three-dimensional epidermis models showed that host antimicrobial peptide expression was induced by SE but not SA. Protein analysis of bacterial cocultured models showed that SA exposure induced inflammatory mediator expression, indicating keratinocyte activation of other epidermal immune populations. Single-cell DropSeq analysis of unchallenged naive, SE-challenged and SA-challenged epidermis models was undertaken to distinguish cells from basal, spinous and granular layers, and to interrogate them in relation to model exposure. In contrast to SE, SA specifically induced a subpopulation of spinous cells that highly expressed transcripts related to epidermal inflammation and antimicrobial response. Furthermore, SA, but not SE, specifically induced a basal population that highly expressed interleukin-1 alarmins. Conclusions These findings suggest that SA-associated remodelling of the epidermis is compartmentalized to different keratinocyte populations. Elucidating the mechanisms regulating bacterial sensing-triggered inflammatory responses within tissues will enable further understanding of microbiome dysbiosis and inflammatory skin diseases, such as atopic eczema.

Keratinocytes produce lipids that are critical for the skin barrier, however, little is known about the impact of age on fatty acid (FA) biosynthesis in these cells. We have examined the relationship between keratinocyte FA composition, lipid biosynthetic gene expression, gene promoter methylation and age. Expression of elongase (ELOVL6 and 7) and desaturase (FADS1 and 2) genes was lower in adult versus neonatal keratinocytes, and was associated with lower concentrations of n-7, n-9 and n-10 polyunsaturated FA in adult cells. Consistent with these findings, transient FADS2 knockdown in neonatal keratinocytes mimicked the adult keratinocyte FA profile in neonatal cells. Interrogation of methylation levels across the FADS2 locus (53 genomic sites) revealed differential methylation of 15 sites in neonatal versus adult keratinocytes, of which three hypermethylated sites in adult keratinocytes overlapped with a SMARCA4 protein binding site in the FADS2 promoter.

Among various cutaneous disorders, acne is often accompanied by post inflammatory hyperpigmentation (PIH). Such increases in pigmentation are controlled not only by secreted factors from keratinocytes, but also from dermal fibroblasts which respond to inflammatory stimuli from the epidermis. Niacinamide is a cosmetic ingredient with proven clinical efficacy against acne-associated PIH. In this study we evaluated the role of Serpin Family B Member 3 (SERPINB3), an endogenous protease inhibitor reported to be upregulated in acne lesions as a potential regulator of the PIH response and its modulation by niacinamide. Analysis of congruent gene expression data from different cell types revealed that while skin keratinocytes expressed high levels of SERPINB3 mRNA, unstimulated fibroblasts expressed negligible amounts. However, treatment of primary human dermal fibroblasts with interleukin-1α (IL-1α) significantly induced SERPINB3 expression and introduction of niacinamide controlled this effect, thereby highlighting its modulatory effect on SERPINB3. Interestingly, Ingenuity Pathway Analysis–based studies revealed possible linkages between SERPINB3 and skin pigmentation. Treatment of primary human melanocytes with recombinant SERPINB3 increased cellular melanin content in a dose-dependent manner, confirming the in silico findings and indicating a potential role for SERPINB3 in skin pigmentation. Its heightened expression in fibroblasts in response to a proinflammatory stimulus such as IL-1α, together with its promelanogenic potential raises the possibility that SERPINB3 may be a stress response paracrine factor deployed to increase epidermal pigmentation. The ability to blunt its expression, provides novel insights into potential mechanisms underlying the anti-inflammatory and anti-PIH activity of niacinamide for amelioration of acne.

BACKGROUND:Atopic dermatitis (AD) arises from a complex interaction between an impaired epidermal barrier, environmental exposures, and the infiltration of T helper (Th)1/Th2/Th17/Th22 T cells. Transcriptomic analysis has advanced our understanding of gene expression in cells and tissues. However, molecular quantitation of cytokine transcripts does not predict the importance of a specific pathway in AD or cellular responses to different inflammatory stimuli. OBJECTIVES:To understand changes in keratinocyte transcriptomic programmes in human cutaneous disease during development of inflammation and in response to treatment. METHODS:We performed in silico deconvolution of the whole-skin transcriptome. Using co-expression clustering and machine-learning tools, we resolved the gene expression of bulk skin (seven datasets, n = 406 samples), firstly, into keratinocyte phenotypes identified by unsupervised clustering and, secondly, into 19 cutaneous cell signatures of purified populations from publicly available datasets. RESULTS:We identify three unique transcriptomic programmes in keratinocytes - KC1, KC2 and KC17 - characteristic of immune signalling from disease-associated Th cells. We cross-validate those signatures across different skin inflammatory conditions and disease stages and demonstrate that the keratinocyte response during treatment is therapy dependent. Broad-spectrum treatment with ciclosporin ameliorated the KC17 response in AD lesions to a nonlesional immunophenotype, without altering KC2. Conversely, the specific anti-Th2 therapy, dupilumab, reversed the KC2 immunophenotype. CONCLUSIONS:Our analysis of transcriptomic signatures in cutaneous disease biopsies reveals the effect of keratinocyte programming in skin inflammation and suggests that the perturbation of a single axis of immune signal alone may be insufficient to resolve keratinocyte immunophenotype abnormalities.

The in situ control of redox insult in human organs is of major clinical relevance, yet remains incompletely understood. Activation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), the "master regulator" of genes controlling cellular redox homeostasis, is advocated as a therapeutic strategy for diseases with severely impaired redox balance. It remains to be shown whether this strategy is effective in human organs, rather than only in isolated human cell types. We have therefore explored the role of Nrf2 in a uniquely accessible human (mini-) organ: scalp hair follicles. Microarray and qRT-PCR analysis of human hair follicles after Nrf2 activation using sulforaphane identified the modulation of phase II metabolism, reactive oxygen species clearance, the pentose phosphate pathway, and glutathione homeostasis. Nrf2 knockdown (small interfering RNA) in cultured human hair follicles confirmed the regulation of key Nrf2 target genes (i.e., heme oxygenase-1, NAD(P)H dehydrogenase, quinone 1, glutathione reductase, glutamate-cysteine ligase catalytic subunit, ABCC1, peroxiredoxin 1). Importantly, Nrf2 activation significantly reduced reactive oxygen species levels and associated lipid peroxidation. Nrf2 preactivation reduced premature catagen and hair growth inhibition induced by oxidative stress (H2O2 or menadione), significantly ameliorated the H2O2-dependent increase in matrix keratinocyte apoptosis and reversed the reactive oxygen species-induced reduction in hair matrix proliferation. This study thus provides direct evidence for the crucial role of Nrf2 in protecting human organ function (i.e., scalp hair follicles) against redox insult.

Sorghum bicolor is an important cereal crop grown on the arid and semi-arid regions of >98 different countries. These regions are such that this crop is often subjected to low water conditions, which can compromise yields. Stay-green sorghum plants are able to retain green leaf area for longer under drought conditions and as such have higher yields than their senescent counterparts. However, the molecular and physiological basis of this drought tolerance is yet to be fully understood. Here, a transcriptomic approach was used to compare gene expression between stay-green (B35) and senescent (R16) sorghum varieties. Ontological analysis of the differentially expressed transcripts identified an enrichment of genes involved with the 'response to osmotic stress' Gene Ontology (GO) category. In particular, delta1-pyrroline-5-carboxylate synthase 2 (P5CS2) was highly expressed in the stay-green line compared with the senescent line, and this high expression was correlated with higher proline levels. Comparisons of the differentially expressed genes with those that lie in known stay-green qualitative trait loci (QTLs) revealed that P5CS2 lies within the Stg1 QTL. Polymorphisms in known cis-elements were identified in the putative promoter region of P5CS2 and these could be responsible for the differences in the expression of this gene. This study provides greater insight into the stay-green trait in sorghum. This will be greatly beneficial not only to improve our understanding of drought tolerance mechanisms in sorghum, but also to facilitate the improvement of future sorghum cultivars by marker-assisted selection (MAS).

Background: Abiotic stresses which include drought and heat are amongst the main limiting factors for plant growth and crop productivity. In the field, these stress types are rarely presented individually and plants are often subjected to a combination of stress types. Sorghum bicolor is a cereal crop which is grown in arid and semi-arid regions and is particularly well adapted to the hot and dry conditions in which it originates and is now grown as a crop. In order to better understand the mechanisms underlying combined stress tolerance in this important crop, we have used microarrays to investigate the transcriptional response of Sorghum subjected to heat and drought stresses imposed both individually and in combination. Results: Microarrays consisting of 28585 gene probes identified gene expression changes equating to ~4% and 18% of genes on the chip following drought and heat stresses respectively. In response to combined stress ~20% of probes were differentially expressed. Whilst many of these transcript changes were in common with those changed in response to heat or drought alone, the levels of 2043 specific transcripts (representing 7% of all gene probes) were found to only be changed following the combined stress treatment. Ontological analysis of these ‘unique’ transcripts identified a potential role for specific transcription factors including MYB78 and ATAF1, chaperones including unique heat shock proteins (HSPs) and metabolic pathways including polyamine biosynthesis in the Sorghum combined stress response. Conclusions: These results show evidence for both cross-talk and specificity in the Sorghum response to combined heat and drought stress. It is clear that some aspects of the combined stress response are unique compared to those of individual stresses. A functional characterization of the genes and pathways identified here could lead to new targets for the enhancement of plant stress tolerance, which will be particularly important in the face of climate change and the increasing prevalence of these abiotic stress types.

Dandruff is a troubling consumer problem characterized by flaking and pruritus of the scalp and is considered a multifactorial condition with sebum, individual susceptibility and the fungus Malassezia all thought to play a part. The condition is commonly treated with shampoo products containing antifungal ingredients such as zinc pyrithione and climbazole. It is hypothesized that these ingredients may be delivering additional scalp skin benefits besides their antifungal activity helping to relieve dandruff effectively. The objective of this study was to evaluate the anti‐dandruff ingredient climbazole for potential skin benefits using genomics and in vitro assays.

Background Abiotic stresses which include drought and heat are amongst the main limiting factors for plant growth and crop productivity. In the field, these stress types are rarely presented individually and plants are often subjected to a combination of stress types. Sorghum bicolor is a cereal crop which is grown in arid and semi-arid regions and is particularly well adapted to the hot and dry conditions in which it originates and is now grown as a crop. In order to better understand the mechanisms underlying combined stress tolerance in this important crop, we have used microarrays to investigate the transcriptional response of Sorghum subjected to heat and drought stresses imposed both individually and in combination. Results Microarrays consisting of 28585 gene probes identified gene expression changes equating to ~4% and 18% of genes on the chip following drought and heat stresses respectively. In response to combined stress ~20% of probes were differentially expressed. Whilst many of these transcript changes were in common with those changed in response to heat or drought alone, the levels of 2043 specific transcripts (representing 7% of all gene probes) were found to only be changed following the combined stress treatment. Ontological analysis of these ‘unique’ transcripts identified a potential role for specific transcription factors including MYB78 and ATAF1, chaperones including unique heat shock proteins (HSPs) and metabolic pathways including polyamine biosynthesis in the Sorghum combined stress response. Conclusions These results show evidence for both cross-talk and specificity in the Sorghum response to combined heat and drought stress. It is clear that some aspects of the combined stress response are unique compared to those of individual stresses. A functional characterization of the genes and pathways identified here could lead to new targets for the enhancement of plant stress tolerance, which will be particularly important in the face of climate change and the increasing prevalence of these abiotic stress types.

Senescence is thought to play an important role in the progressive age-related decline in tissue integrity and concomitant diseases, but not much is known about the complex interplay between upstream regulators and downstream effectors. We profiled whole genome gene expression of non-stressed and rotenone-stressed human fibroblast strains from young and oldest old subjects, and measured senescence associated β-gal activity. Microarray results identified gene sets involved in carbohydrate metabolism, Wnt/β-catenin signaling, the cell cycle, glutamate signaling, RNA-processing and mitochondrial function as being differentially regulated with chronological age. The most significantly differentially regulated mRNA corresponded to the p16 gene. p16 was then investigated using qPCR, Western blotting and immunocytochemistry. In conclusion, we have identified cellular pathways that are differentially expressed between fibroblast strains from young and old subjects.

The effect of hypoxia on global gene expression in human adipocytes has been examined using DNA microarrays. Adipocytes (Zen-Bio, day 12 post-differentiation) were exposed to hypoxia (1% O(2)) or 'normoxia' (21% O(2)) for 24 h and extracted RNA probed with Agilent arrays containing 41,152 probes. A total of 1346 probes were differentially expressed (>2.0-fold change, P < 0.01) in response to hypoxia; 650 genes were up-regulated (including LEP, IL6, VEGF, ANGPTL4) and 650 down-regulated (including ADIPOQ, UCP2). Major genes not previously identified as hypoxia-sensitive in adipocytes include AQP3, FABP3, FABP5 and PPARGC1A. Ingenuity analysis indicated that several pathways and functions were modulated by hypoxia, including glucose utilization, lipid oxidation and cell death. Network analysis indicated a down-regulation of p38/MAPK and PGC-1α signalling in the adipocytes. It is concluded that hypoxia has extensive effects on human adipocyte gene expression, consistent with low O(2) tension underlying adipose tissue dysfunction in obesity.

Natural human skin colour is determined both by environmental exposure to ultraviolet light and through inherited genetic variation in a very limited number of genes. Variation of a non-synonymous single-nucleotide polymorphism (nsSNP; rs1426654) in the gene (SLC24A5) encoding the NCKX5 protein is associated with differences in constitutive skin colour in South Asians. The nsSNP encodes the substitution of alanine for threonine at residue 111 (A111T) near a transmembrane region required for exchanger activity, a region which is highly conserved across different species and between NCKX family members. We have shown that NCKX5 is located at the trans-Golgi network of melanocytes and functions as a potassium-dependent sodium-calcium exchanger. When heterologously expressed, the 111T variant of NCKX5 shows significantly lower exchanger activity than the A111 variant. We have postulated that lower exchanger activity causes the reduced melanogenesis and lighter skin in Thr111-positive individuals. We used gene expression microarrays with qPCR replication and validation to assess the impact of siRNA-mediated knockdown of SLC24A5 on the transcriptome of cultured normal human melanocytes (NHM). Very few genes associated with melanogenesis were altered at the transcript level except for MC1R, suggesting that SLC24A5 interacts with at least one well-characterized melanogenic signalling pathway. More surprisingly, the expression of a number of cholesterol homeostatic genes was altered after SLC24A5 knockdown, and the total cholesterol content of NHM was increased. Cholesterol has previously been identified as a potential melanogenic regulator, and our data imply that NCKX5 exchanger function influences natural variation in skin pigmentation via a novel, unknown mechanism affecting cellular sterol levels.

The aim of this study was to examine the effects of macrophage secretions on global gene expression in human preadipocytes using microarrays. Preadipocytes were cultured with unconditioned or conditioned medium from U937 macrophages, and gene expression examined with Agilent arrays (43,000 probes). 472 transcripts were differentially regulated (>2-fold difference; P<0.05) between preadipocytes in the conditioned medium compared to the unconditioned; 401 were upregulated and 71 downregulated. The upregulated transcripts were particularly linked to inflammation, including IL-1β, IL-6, and CCL20 (16.8-, 10.0-, and 8.9-fold increases, respectively) together with matrix metalloproteinases (MMP3, MMP9 and MMP12). Major pathways regulated by the conditioned medium were linked to inflammation, macrophage infiltration and lipid accumulation. Network analysis identified NFkB and IL-1β as central nodes in the upregulation of multiple inflammation-related genes. Treatment with an IL-1β neutralising antibody abolished the stimulation of IL-6 secretion by conditioned medium, indicating that IL-1β is a key regulator of preadipocyte IL-6 production. Macrophages evoke extensive changes in preadipocyte gene expression.

In vitro organ culture can provide insight into isolated mucosal responses to particular environmental stimuli. The objective of the present study was to investigate the impact of a prolonged culturing time as well as the addition of acidic gastric fluid into the in vitro environment of cultured gastric antral tissue to evaluate how altering the commonly used neutral environment impacted tissue. Furthermore, we aimed to investigate the impact of G's Formula, a dietary supplement for horses, on the secretion of gastrin, interleukin1-beta (IL-1β), and nitric oxide (NO). These biomarkers are of interest due to their effects on gastric motility and mucosal activity. Gastric mucosal tissue explants from porcine stomachs were cultured in the presence of a simulated gastric fluid (BL, n = 14), simulated gastric fluid containing the dietary supplement G's Formula (DF, n = 12), or an equal volume of phosphate buffered saline (CO, n = 14). At 48 and 60 h, 10−5 M carbachol was used to stimulate gastrin secretion. Cell viability was assessed at 72 h using calcein and ethidium-homodimer 1 staining. Media was analyzed for gastrin, IL-1β, and NO at 48, 60, and 72 h. There were no effects of treatment or carbachol stimulation on explant cell viability. Carbachol resulted in a significant increase in gastrin concentration in CO and DF treatments, but not in BL. NO was higher in CO than in BL, and NO increased in the CO and DF treatments but not in BL. In conclusion, the addition of carbachol and gastric digests to culture media did not impact cell viability. The use of an acidic gastric digest (BL) reduced the effect of cholinergic stimulation with carbachol at a concentration of 10−5 M and reduced NO secretion. The addition of the dietary supplement to the gastric digest (DF) appeared to mediate these effects within this model. Further research is required to evaluate the specific effects of this dietary supplement on direct markers of mucosal activity and the functional relevance of these results in vivo.