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Keratinocytes are the most abundant cell type in the epidermis. They prevent desiccation and provide immunological and barrier defense against potential pathogens such as Staphylococcus aureus and Candida albicans. The study of this first line of immune defense may be hindered by invasive isolation methods and/or improper culture conditions to support stem cell maintenance and other potential mechanisms contributing to long-term subcultivation in vitro. Primary keratinocytes have been successfully isolated from blister roofs induced by negative pressure, which separates the epidermis from the dermis in vivo in human subjects. This method allows collection of pure epidermal cells without dermal contamination in a minimally invasive manner. However, the isolated keratinocytes differentiate and senesce when cultured in vitro beyond five passages. Here, we present evidence that the Rho kinase (ROCK) inhibitor Y-27632 can be used to effectively increase the proliferative capabilities of keratinocytes isolated using the suction blister method, similar to what has been previously reported for primary keratinocytes isolated using alternative methods. We show that the increase in passage number is directly correlated to delayed differentiation, and that cells passaged long term with the inhibitor retain their ability to stratify in organotypic raft cultures and respond to cytokine treatment; additionally, the late passage cells have a heterogeneous mix of differentiated and non-differentiated cells which may be predicted by a ratio of select differentiation markers. The described method presents a minimally invasive procedure for keratinocyte isolation and prolonged culture that allows analysis of keratinocyte function in both healthy volunteers and patients with dermatologic diseases. [ABSTRACT FROM AUTHOR]

The “Western diet” is characterized by increased intake of saturated and omega-6 (n−6) fatty acids with a relative reduction in omega-3 (n−3) consumption. These fatty acids can directly and indirectly modulate the gut microbiome, resulting in altered host immunity. Omega-3 fatty acids can also directly modulate immunity through alterations in the phospholipid membranes of immune cells, inhibition of n−6 induced inflammation, down-regulation of inflammatory transcription factors, and by serving as pre-cursors to anti-inflammatory lipid mediators such as resolvins and protectins. We have previously shown that consumption by breeder mice of diets high in saturated and n−6 fatty acids have inflammatory and immune-modulating effects on offspring that are at least partially driven by vertical transmission of altered gut microbiota. To determine if parental diets high in n−3 fatty acids could also affect offspring microbiome and immunity, we fed breeding mice an n−3-rich diet with 40% calories from fat and measured immune outcomes in their offspring. We found offspring from mice fed diets high in n−3 had altered gut microbiomes and modestly enhanced anti-inflammatory IL-10 from both colonic and splenic tissue. Omega-3 pups were protected during peanut oral allergy challenge with small but measurable alterations in peanut-related serologies. However, n−3 pups displayed a tendency toward worsened responses during E. coli sepsis and had significantly worse outcomes during Staphylococcus aureus skin infection. Our results indicate excess parental n−3 fatty acid intake alters microbiome and immune response in offspring. [ABSTRACT FROM AUTHOR]

Helicobacter pylori infection is characterized by infiltration of cells of the immune system, including dendritic cells, into the gastric mucosa. During chronic inflammation with Helicobacter pylori infection, a variety of cytokines are secreted into the mucosa, including interleukin-1β (IL-1β). The role of IL-1 in H. pylori infection was investigated using bone-marrow-derived dendritic cells from wild-type and IL-1 receptor-deficient (IL-1R−/−) mice. Dendritic cells were incubated with H. pylori at a multiplicity of infection of 10 and 100, and cytokine production evaluated. Helicobacter pylori SS1, H. pylori SD4, and an isogenic cagE mutant of SD4 stimulated IL-12, IL-6, IL-1β, IL-10, and tumor necrosis factor-alpha at comparable levels in dendritic cells from both wild-type and IL-1R−/− mice. IL-10 production required the higher inoculum, while IL-12 was decreased at this bacterial load. Pretreatment of dendritic cells with an antibody to IL-10 resulted in an increased production of IL-12, confirming the down-regulation of IL-12 by IL-10. cagE was required for maximum stimulation of IL-12 by H. pylori. We speculate that the down-regulation of IL-12 by IL-10 at the higher multiplicity of infection represents the modulation of the host inflammatory response in vivo by H. pylori when the bacterial load is high, allowing for persistent colonization of the gastric mucosa. [ABSTRACT FROM AUTHOR]