Schlafen 3 knockout mice display gender-specific differences in weight gain, food efficiency, and expression of markers of intestinal epithelial differentiation, metabolism, and immune cell function

Self-renewal and differentiation are essential for intestinal epithelium absorptive functioning and adaptation to pathological states such as short gut syndrome, ulcers, and inflammatory bowel disease. The rodent Slfn3 and its human analog Slfn12 are critical in regulating intestinal epithelial differentiation. We sought to characterize intestinal function in Slfn3 knockout (KO) mice. Male and female pair-fed Slfn3KO mice gained less weight with decreased food efficiency than wild type (WT) mice, with more pronounced effects in females. RNA sequencing performed on intestinal mucosa of Slfn3KO and WT mice showed gene ontology decreases in cell adhesion molecule signaling, tumor necrosis factor receptor binding, and adaptive immune cell proliferation/functioning genes in Slfn3KO mice, with greater effects in females. qPCR analysis of fatty acid metabolism genes, Pla2g4c, Pla2g2f, and Cyp3c55 revealed an increase in Pla2g4c, and a decrease in Pla2g2f in Slfn3KO females. Additionally, adipogenesis genes, Fabp4 and Lpl were decreased and ketogenesis gene Hmgcs2 was increased in female Slfn3KO mice. Sequencing did not reveal significant changes in differentiation markers, so qPCR was utilized. Slfn3KO tended to have decreased expression of intestinal differentiation markers sucrase isomaltase, dipeptidyl peptidase 4, villin 1, and glucose transporter 1 (Glut1) vs. WT males, although these trends did not achieve statistical significance unless data from several markers was pooled. Differentiation markers, Glut2 and sodium-glucose transporter 1 (SGLT1), did show statistically significant sex-dependent differences. Glut2 mRNA was reduced in Slfn3KO females, while SGLT1 increased in Slfn3KO males. Notch2 and Cdx2 were only increased in female Slfn3KO mice. Although Slfn3KO mice gain less weight and decreased food efficiency, their biochemical phenotype is more subtle and suggests a complex interplay between gender effects, Slfn3, and another regulatory pathway yet to be identified that compensates for the chronic loss of Slfn3

Gene expression profiling and network analysis of peripheral blood monocytes in a chronic model of allergic asthma

The Aspergillus fumigatus mouse model of asthma mimics the characteristics of human fungal asthma, including local and systemic inflammation. Monocyte/macrophage lineage cells direct innate immune responses and guide adaptive responses. To identify gene expression changes in peripheral blood monocytes in the context of fungal allergy, mice were exposed to systemic and intranasal inoculations of fungal antigen (sensitized), and naïve and sensitized animals were challenged intratracheally with live A. fumigatus conidia. Microarray analysis of blood monocytes from allergic versus non-allergic mice showed ≥ twofold modulation of 45 genes. Ingenuity pathway analysis revealed a network of these genes involved in antigen presentation, inflammation, and immune cell trafficking. These data show that allergen sensitization and challenge affects gene expression in peripheral monocytes.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/79085/1/j.1348-0421.2010.00242.x.pd

Schlafen 12 Interaction with SerpinB12 and Deubiquitylases Drives Human Enterocyte Differentiation

Background/Aims: Human enterocytic differentiation is altered during development, fasting, adaptation, and bariatric surgery, but its intracellular control remains unclear. We hypothesized that Schlafen 12 (SLFN12) regulates enterocyte differentiation. Methods: We used laser capture dissection of epithelium, qRT-PCR, and immunohistochemistry to evaluate SLFN12 expression in biopsies of control and fasting human duodenal mucosa, and viral overexpression and siRNA to trace the SLFN12 pathway in human Caco-2 and HIEC6 intestinal epithelial cells. Results: Fasting human duodenal mucosa expressed less SLFN12 mRNA and protein, accompanied by decreases in enterocytic markers like sucrase-isomaltase. SLFN12 overexpression increased Caco-2 sucrase-isomaltase promoter activity, mRNA, and protein independently of proliferation, and activated the SLFN12 putative promoter. SLFN12 coprecipitated Serpin B12 (SERPB12). An inactivating SLFN12 point mutation prevented both SERPB12 binding and sucrase-isomaltase induction. SERPB12 overexpression also induced sucrase-isomaltase, while reducing SERPB12 prevented the SLFN12 effect on sucrase-isomaltase. Sucrase-isomaltase induction by both SLFN12 and SERPB12 was attenuated by reducing UCHL5 or USP14, and blocked by reducing both. SERPB12 stimulated USP14 but not UCHL5 activity. SERPB12 coprecipitated USP14 but not UCHL5. Moreover, SLFN12 increased protein levels of the sucrase-isomaltase-promoter-binding transcription factor cdx2 without altering Cdx2 mRNA. This was prevented by reducing UCHL5 and USP14. We further validated this pathway in vitro and in vivo. SLFN12 or SERPB12 overexpression induced sucrase-isomaltase in human non-malignant HIEC-6 enterocytes. Conclusions: SLFN12 regulates human enterocytic differentiation by a pathway involving SERPB12, the deubiquitylases, and Cdx2. This pathway may be targeted to manipulate human enterocytic differentiation in mucosal atrophy, short gut or obesity

Regulatory iNKT cells lack PLZF expression and control Treg cell and macrophage homeostasis in adipose tissue

iNKT cells are CD1d-restricted lipid-sensing innate T cells that express the transcription factor PLZF. iNKT cells accumulate in adipose tissue, where they are anti-inflammatory, but the factors that contribute to their anti-inflammatory nature, and their targets in adipose tissue are unknown. Here we report that adipose tissue iNKT cells have a unique transcriptional program and produce interleukin 2 (IL-2) and IL-10. Unlike other iNKT cells, they lack PLZF, but express the transcription factor E4BP4, which controls their IL-10 production. Adipose iNKT cells are a tissue resident population that induces an anti-inflammatory phenotype in macrophages and, through production of IL-2, controls the number, proliferation and suppressor function of adipose regulatory T (Treg) cells. Thus, adipose tissue iNKT cells are unique regulators of immune homeostasis in this tissue

Loss of Schlafen3 influences the expression levels of Schlafen family members in ileum, thymus, and spleen tissue

Background The Schlafen (Slfn) family proteins are important for regulation of cell growth, cell differentiation and cell cycle progression. We sought to distinguish Slfn family expression in Slfn3 knockout (KO) mice after RNA sequencing analysis of Slfn3KO vs. wildtype (WT) mice revealed varying expressions of Slfn family in ileal mucosa. Methods Quantitative PCR analysis of Slfn members was evaluated in ileal mucosa, thymus and spleen tissue since Slfn family members have roles in differentiating intestinal and immune cells. Results Ileal mucosa of Slfn3KO mice displayed a decrease in Slfn3, 4, 8 and 9 while Slfn1 and 5 increased in mRNA expression vs. WT mice. Thymic tissue had a Slfn9 increase and a Slfn4 decrease while splenic tissue had a Slfn8 and Slfn9 increase in Slfn3KO mice vs. WT mice. These differential expressions of Slfn members could indicate a feedback regulatory mechanism within the Slfn family. Indeed, MATCH™ tool from geneXplain predicted that all Slfn members have regions in their promoters for the Kruppel-like factor-6 transcription factor. In addition, NFAT related factors, ING4, ZNF333 and KLF4 are also predicted to bind in up to 6 of the 8 Slfn promoters. This study further describes a possible autoregulatory mechanism amongst the Slfn family members which could be important in how they regulate the differentiation of various cell types

Vil-Cre specific Slfn3KO mice exhibit sex-specific differences in lung, stomach, cecum, kidney, and proximal colon differentiation markers and Slfn family members expression levels

The Schlafen (Slfn) family proteins are critical regulators of cell proliferation, induction of immune responses, differentiation, self-restoration, and cell cycle progression. Rodent Slfn3 and human ortholog SLFN12 are critical in the regulation of intestinal epithelial differentiation. Following previous work utilizing Vil-Cre epithelial-specific Slfn3 knockout (VC-Slfn3KO) mice to evaluate Slfn3's role in small intestinal epithelial differentiation, we sought to characterize and distinguish the effects of Slfn3 loss on Slfn family member mRNA expression and differentiation markers for other epithelial cells in the lung, stomach, cecum, and proximal colon. Quantitative PCR analysis of Slfn1, 2, 4, 5, 8, and 9 and multiple differentiation markers were evaluated. We observed gender-specific effects with the loss of Slfn3 on the other Slfn family members and epithelial differentiation markers expression. Lung Slfn4 and 5 were increased only in male VC-Slfn3KO while lung Slfn2 and 8 were decreased only in female VC-Slfn3KO compared to controls. Slfn1, 2, 4, and 9 were increased in the gastric mucosa of male VC-Slfn3KO mice compared to controls. Slfn5 was reduced in female VC-Slfn3KO proximal colonic mucosa compared to controls. Lung AT1 cell differentiation marker Hopx mRNA expression was decreased and Ager was increased in VC-Slfn3KO male mice compared to controls. Lung AT2 differentiation markers and surfactant genes Sftpc and Sftpd were decreased in male VC-Slfn3KO mice. Stomach transcription factors, Lgr5 and Notch1 were increased in male VC-Slfn3KO. Tff1 secretory protein gene was decreased in female VC-Slfn3KO mice. Sucrase isomaltase was greatly increased in male VC-Slfn3KO mice in both cecal and proximal colonic mucosa, but glucose transporter Glut2 was decreased only in the cecum of female VC-Slfn3KO. The changes induced by VC-Slfn3KO in the expression of epithelial differentiation markers and other Schlafen proteins in various target tissues, indicate a complex regulation of gene expression that is sex-dependent

Schlafen 3 knockout mice display gender-specific differences in weight gain, food efficiency, and expression of markers of intestinal epithelial differentiation, metabolism, and immune cell function.

Self-renewal and differentiation are essential for intestinal epithelium absorptive functioning and adaptation to pathological states such as short gut syndrome, ulcers, and inflammatory bowel disease. The rodent Slfn3 and its human analog Slfn12 are critical in regulating intestinal epithelial differentiation. We sought to characterize intestinal function in Slfn3 knockout (KO) mice. Male and female pair-fed Slfn3KO mice gained less weight with decreased food efficiency than wild type (WT) mice, with more pronounced effects in females. RNA sequencing performed on intestinal mucosa of Slfn3KO and WT mice showed gene ontology decreases in cell adhesion molecule signaling, tumor necrosis factor receptor binding, and adaptive immune cell proliferation/functioning genes in Slfn3KO mice, with greater effects in females. qPCR analysis of fatty acid metabolism genes, Pla2g4c, Pla2g2f, and Cyp3c55 revealed an increase in Pla2g4c, and a decrease in Pla2g2f in Slfn3KO females. Additionally, adipogenesis genes, Fabp4 and Lpl were decreased and ketogenesis gene Hmgcs2 was increased in female Slfn3KO mice. Sequencing did not reveal significant changes in differentiation markers, so qPCR was utilized. Slfn3KO tended to have decreased expression of intestinal differentiation markers sucrase isomaltase, dipeptidyl peptidase 4, villin 1, and glucose transporter 1 (Glut1) vs. WT males, although these trends did not achieve statistical significance unless data from several markers was pooled. Differentiation markers, Glut2 and sodium-glucose transporter 1 (SGLT1), did show statistically significant sex-dependent differences. Glut2 mRNA was reduced in Slfn3KO females, while SGLT1 increased in Slfn3KO males. Notch2 and Cdx2 were only increased in female Slfn3KO mice. Although Slfn3KO mice gain less weight and decreased food efficiency, their biochemical phenotype is more subtle and suggests a complex interplay between gender effects, Slfn3, and another regulatory pathway yet to be identified that compensates for the chronic loss of Slfn3

ZINC40099027 activates human focal adhesion kinase by accelerating the enzymatic activity of the FAK kinase domain

Abstract Focal adhesion kinase (FAK) regulates gastrointestinal epithelial restitution and healing. ZINC40099027 (Zn27) activates cellular FAK and promotes intestinal epithelial wound closure in vitro and in mice. However, whether Zn27 activates FAK directly or indirectly remains unknown. We evaluated Zn27 potential modulation of the key phosphatases, PTP‐PEST, PTP1B, and SHP2, that inactivate FAK, and performed in vitro kinase assays with purified FAK to assess direct Zn27‐FAK interaction. In human Caco‐2 cells, Zn27‐stimulated FAK‐Tyr‐397 phosphorylation despite PTP‐PEST inhibition and did not affect PTP1B‐FAK interaction or SHP2 activity. Conversely, in vitro kinase assays demonstrated that Zn27 directly activates both full‐length 125 kDa FAK and its 35 kDa kinase domain. The ATP‐competitive FAK inhibitor PF573228 reduced basal and ZN27‐stimulated FAK phosphorylation in Caco‐2 cells, but Zn27 increased FAK phosphorylation even in cells treated with PF573228. Increasing PF573228 concentrations completely prevented activation of 35 kDa FAK in vitro by a normally effective Zn27 concentration. Conversely, increasing Zn27 concentrations dose‐dependently activated kinase activity and overcame PF573228 inhibition of FAK, suggesting the direct interactions of Zn27 with FAK may be competitive. Zn27 increased the maximal activity (Vmax) of FAK. The apparent Km of the substrate also increased under laboratory conditions less relevant to intracellular ATP concentrations. These results suggest that Zn27 is highly potent and enhances FAK activity via allosteric interaction with the FAK kinase domain to increase the Vmax of FAK for ATP. Understanding Zn27 enhancement of FAK activity will be important to redesign and develop a clinical drug that can promote mucosal wound healing

A murine model of Lyme disease demonstrates that Borrelia burgdorferi colonizes the dura mater and induces inflammation in the central nervous system.

Lyme disease, which is caused by infection with Borrelia burgdorferi and related species, can lead to inflammatory pathologies affecting the joints, heart, and nervous systems including the central nervous system (CNS). Inbred laboratory mice have been used to define the kinetics of B. burgdorferi infection and host immune responses in joints and heart, however similar studies are lacking in the CNS of these animals. A tractable animal model for investigating host-Borrelia interactions in the CNS is key to understanding the mechanisms of CNS pathogenesis. Therefore, we characterized the kinetics of B. burgdorferi colonization and associated immune responses in the CNS of mice during early and subacute infection. Using fluorescence-immunohistochemistry, intravital microscopy, bacterial culture, and quantitative PCR, we found B. burgdorferi routinely colonized the dura mater of C3H mice, with peak spirochete burden at day 7 post-infection. Dura mater colonization was observed for several Lyme disease agents including B. burgdorferi, B. garinii, and B. mayonii. RNA-sequencing and quantitative RT-PCR showed that B. burgdorferi infection was associated with increased expression of inflammatory cytokines and a robust interferon (IFN) response in the dura mater. Histopathologic changes including leukocytic infiltrates and vascular changes were also observed in the meninges of infected animals. In contrast to the meninges, we did not detect B. burgdorferi, infiltrating leukocytes, or large-scale changes in cytokine profiles in the cerebral cortex or hippocampus during infection; however, both brain regions demonstrated similar changes in expression of IFN-stimulated genes as observed in peripheral tissues and meninges. Taken together, B. burgdorferi is capable of colonizing the meninges in laboratory mice, and induces localized inflammation similar to peripheral tissues. A sterile IFN response in the absence of B. burgdorferi or inflammatory cytokines is unique to the brain parenchyma, and provides insight into the potential mechanisms of CNS pathology associated with this important pathogen

Schlafen 12 Slows TNBC Tumor Growth, Induces Luminal Markers, and Predicts Favorable Survival

The Schlafen 12 (SLFN12) protein regulates triple-negative breast cancer (TNBC) growth, differentiation, and proliferation. SLFN12 mRNA expression strongly correlates with TNBC patient survival. We sought to explore SLFN12 overexpression effects on in vivo human TNBC tumor xenograft growth and performed RNA-seq on xenografts to investigate related SLFN12 pathways. Stable SLFN12 overexpression reduced tumorigenesis, increased tumor latency, and reduced tumor volume. RNA-seq showed that SLFN12 overexpressing xenografts had higher luminal markers levels, suggesting that TNBC cells switched from an undifferentiated basal phenotype to a more differentiated, less aggressive luminal phenotype. SLFN12-overexpressing xenografts increased less aggressive BC markers, HER2 receptors ERBB2 and EGFR expression, which are not detectable by immunostaining in TNBC. Two cancer progression pathways, the NAD signaling pathway and the superpathway of cholesterol biosynthesis, were downregulated with SLFN12 overexpression. RNA-seq identified gene signatures associated with SLFN12 overexpression. Higher gene signature levels indicated good survival when tested on four independent BC datasets. These signatures behaved differently in African Americans than in Caucasian Americans, indicating a possible biological difference between these races that could contribute to the worse survival observed in African Americans with BC. These results suggest an increased SLFN12 expression modulates TNBC aggressiveness through a gene signature that could offer new treatment targets