Smad4 Loss in the Mouse Intestinal Epithelium Alleviates the Pathological Fibrotic Response to Injury in the Colon.

Mucosal healing is associated with better clinical outcomes in patients with inflammatory bowel diseases (IBDs). Unresolved injury and inflammation, on the other hand, increases pathological fibrosis and the predisposition to cancer. Loss of Smad4, a tumor suppressor, is known to increase colitis-associated cancer in mouse models of chronic IBD. Since common biological processes are involved in both injury repair and tumor growth, we sought to investigate the effect of Smad4 loss on the response to epithelial injury. To this end, Smad4 was knocked out specifically in the intestinal epithelium and transcriptomic and morphological changes compared between wild type mice and Smad4 knock out mice after DSS-induced injury. We find that Smad4 loss alleviates pathological fibrosis and enhances mucosal repair. The transcriptomic changes specific to epithelium indicate molecular changes that affect epithelial extracellular matrix (ECM) and promote enhanced mucosal repair. These findings suggest that the biological processes that promote wound healing alleviate the pathological fibrotic response to DSS. Therefore, these mucosal repair processes could be exploited to develop therapies that promote normal wound healing and prevent fibrosis. NEW AND NOTEWORTHY: We show that transcriptomic changes due to Smad4 loss in the colonic epithelium alleviates the pathological fibrotic response to DSS in an IBD mouse model of acute inflammation. Most notably, we find that collagen deposition in the epithelial ECM, as opposed to that in the lamina propria, correlates with epithelial changes that enhance wound healing. This is the first report on a mouse model providing alleviated fibrotic response in a DSS-IBD mouse model in vivo .

Structural modification of C2-substituents on 1,4-bis(arylsulfonamido)benzene or naphthalene-N,N'-diacetic acid derivatives as potent inhibitors of the Keap1-Nrf2 protein-protein interaction.

The Keap1-Nrf2-ARE signaling pathway is an attractive therapeutic target for the prevention and treatment of oxidative stress-associated diseases by activating the cellular expression of cytoprotective enzymes and proteins. Small molecule inhibitors can directly disrupt the Keap1-Nrf2 protein-protein interaction (PPI), resulting in elevated levels of Nrf2 protein and subsequent stimulation of related antioxidant responses. Previously, we found that 1,4-bis(arylsulfonamido)benzene or naphthalene-N,N'-diacetic acid derivatives with an ether type C2-substituent on the benzene or naphthalene core exhibited potent inhibitory activities with IC50's in the submicromolar or nanomolar range. We here describe a more detailed structure-activity relationship study around the C2 substituents containing various polar linkers shedding new insight on their binding interactions with the Keap1 Kelch domain. The key observation from our findings is that the substituents at the C2-position of the benzene or naphthalene scaffold impact their inhibitory potencies in biochemical assays as well as activities in cell culture. The biochemical FP and TR-FRET assays revealed that the naphthalene derivatives 17b and 18 with an additional carboxylate at the C2 were the most active inhibitors against Keap1-Nrf2 PPI. In the cell-based assay, the two compounds were shown to be potent Nrf2 activators of the transcription of the Nrf2-dependent genes, such as HMOX2, GSTM3, and NQO1.

In Vitro Organoid-Based Assays Reveal SMAD4 Tumor-Suppressive Mechanisms for Serrated Colorectal Cancer Invasion.

Colon cancer is the third most prominent cancer and second leading cause of cancer-related deaths in the United States. Up to 20% of colon cancers follow the serrated tumor pathway driven by mutations in the MAPK pathway. Loss of SMAD4 function occurs in the majority of late-stage colon cancers and is associated with aggressive cancer progression. Therefore, it is important to develop technology to accurately model and better understand the genetic mechanisms behind cancer invasion. Organoids derived from tumors found in the Smad4KO BRAFV600E/+ mouse model present multiple phenotypes characteristic of invasion both in ex vivo and in vivo systems. Smad4KO BRAFV600E/+ tumor organoids can migrate through 3D culture and infiltrate through transwell membranes. This invasive behavior can be suppressed when SMAD4 is re-expressed in the tumor organoids. RNA-Seq analysis reveals that SMAD4 expression in organoids rapidly regulates transcripts associated with extracellular matrix and secreted proteins, suggesting that the mechanisms employed by SMAD4 to inhibit invasion are associated with regulation of extracellular matrix and secretory pathways. These findings indicate new models to study SMAD4 regulation of tumor invasion and an additional layer of complexity in the tumor-suppressive function of the SMAD4/Tgfß pathway.

TGFB1 induces fetal reprogramming and enhances intestinal regeneration.

The gut epithelium has a remarkable ability to recover from damage. We employed a combination of high-throughput sequencing approaches, mouse genetics, and murine and human organoids and identified a role for TGFB signaling during intestinal regeneration following injury. At 2 days following irradiation (IR)-induced damage of intestinal crypts, a surge in TGFB1 expression is mediated by monocyte/macrophage cells at the location of damage. The depletion of macrophages or genetic disruption of TGFB signaling significantly impaired the regenerative response. Intestinal regeneration is characterized by the induction of a fetal-like transcriptional signature during repair. In organoid culture, TGFB1 treatment was necessary and sufficient to induce the fetal-like/regenerative state. Mesenchymal cells were also responsive to TGFB1 and enhanced the regenerative response. Mechanistically, pro-regenerative factors, YAP/TEAD and SOX9, are activated in the epithelium exposed to TGFB1. Finally, pre-treatment with TGFB1 enhanced the ability of primary epithelial cultures to engraft into damaged murine colon, suggesting promise for cellular therapy.

Multiple roles and regulatory mechanisms of the transcription factor HNF4 in the intestine.

Hepatocyte nuclear factor 4-alpha (HNF4α) drives a complex array of transcriptional programs across multiple organs. Beyond its previously documented function in the liver, HNF4α has crucial roles in the kidney, intestine, and pancreas. In the intestine, a multitude of functions have been attributed to HNF4 and its accessory transcription factors, including but not limited to, intestinal maturation, differentiation, regeneration, and stem cell renewal. Functional redundancy between HNF4α and its intestine-restricted paralog HNF4γ, and co-regulation with other transcription factors drive these functions. Dysregulated expression of HNF4 results in a wide range of disease manifestations, including the development of a chronic inflammatory state in the intestine. In this review, we focus on the multiple molecular mechanisms of HNF4 in the intestine and explore translational opportunities. We aim to introduce new perspectives in understanding intestinal genetics and the complexity of gastrointestinal disorders through the lens of HNF4 transcription factors.

Hnf4 activates mimetic-cell enhancers to recapitulate gut and liver development within the thymus.

Mimetic cells are medullary thymic epithelial cells (mTECs) that mimic extra-thymic cell types to tolerize T cells to self-antigens. Here, we dissected the biology of entero-hepato mTECs, mimetic cells expressing gut- and liver-associated transcripts. Entero-hepato mTECs conserved their thymic identity yet accessed wide swaths of enterocyte chromatin and transcriptional programs via the transcription factors Hnf4α and Hnf4γ. Deletion of Hnf4α and Hnf4γ in TECs ablated entero-hepato mTECs and downregulated numerous gut- and liver-associated transcripts, with a primary contribution from Hnf4γ. Loss of Hnf4 impaired enhancer activation and CTCF redistribution in mTECs but did not impact Polycomb-mediated repression or promoter-proximal histone marks. By single-cell RNA sequencing, Hnf4 loss produced three distinct effects on mimetic cell state, fate, and accumulation. Serendipitously, a requirement for Hnf4 in microfold mTECs was discovered, which exposed a requirement for Hnf4γ in gut microfold cells and the IgA response. Study of Hnf4 in entero-hepato mTECs thus revealed mechanisms of gene control in the thymus and periphery alike.

ß-Catenin Drives Butyrophilin-like Molecule Loss and γδ T-cell Exclusion in Colon Cancer.

Intraepithelial lymphocytes (IEL) expressing γδ T-cell receptors (γδTCR) play key roles in elimination of colon cancer. However, the precise mechanisms by which progressing cancer cells evade immunosurveillance by these innate T cells are unknown. Here, we investigated how loss of the Apc tumor suppressor in gut tissue could enable nascent cancer cells to escape immunosurveillance by cytotoxic γδIELs. In contrast with healthy intestinal or colonic tissue, we found that γδIELs were largely absent from the microenvironment of both mouse and human tumors, and that butyrophilin-like (BTNL) molecules, which can critically regulate γδIEL through direct γδTCR interactions, were also downregulated in tumors. We then demonstrated that ß-catenin activation through loss of Apc rapidly suppressed expression of the mRNA encoding the HNF4A and HNF4G transcription factors, preventing their binding to promoter regions of Btnl genes. Reexpression of BTNL1 and BTNL6 in cancer cells increased γδIEL survival and activation in coculture assays but failed to augment their cancer-killing ability in vitro or their recruitment to orthotopic tumors. However, inhibition of ß-catenin signaling via genetic deletion of Bcl9/Bcl9L in either Apc-deficient or mutant ß-catenin mouse models restored Hnf4a, Hnf4g, and Btnl gene expression and γδ T-cell infiltration into tumors. These observations highlight an immune-evasion mechanism specific to WNT-driven colon cancer cells that disrupts γδIEL immunosurveillance and furthers cancer progression.

Moringa isothiocyanate-1 inhibits LPS-induced inflammation in mouse myoblasts and skeletal muscle.

This study aims to investigate the anti-inflammatory effects of moringa isothiocyanate-1 (MIC-1) extracted from seeds of Moringa oleifera Lam. in lipopolysaccharide (LPS)-induced inflammation models. MIC-1 decreased nitric oxide production and reduced the expression of pro-inflammatory markers (TNF-α, Ifn-α, IL-1ß, IL-6) in C2C12 myoblasts. The daily oral treatment of MIC-1 (80 mg/kg) for three days significantly reduced the expression of pro-inflammatory markers in gastrocnemius muscle tissue of LPS-treated C57BL/6 male mice. Transcriptomic analysis provided further insights into the inhibitory effects of MIC-1 on the LPS-induced inflammation, which suggested that MIC-1 affects inflammation and immunity-related genes in myoblasts and skeletal muscle tissue. MIC-1 inhibited the nuclear accumulation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in the LPS-treated myoblasts. Our data support the hypothesis that the MIC-1's effects in the muscle cells are mediated through the inhibition of the NF-κB translocation in the nucleus, which, in turn, results in immunomodulating and anti-inflammatory responses at the gene expression levels.

Maf family transcription factors are required for nutrient uptake in the mouse neonatal gut.

There are fundamental differences in how neonatal and adult intestines absorb nutrients. In adults, macromolecules are broken down into simpler molecular components in the lumen of the small intestine, then absorbed. In contrast, neonates are thought to rely on internalization of whole macromolecules and subsequent degradation in the lysosome. Here, we identify the Maf family transcription factors MAFB and c-MAF as markers of terminally differentiated intestinal enterocytes throughout life. The expression of these factors is regulated by HNF4α and HNF4γ, master regulators of enterocyte cell fate. Loss of Maf factors results in a neonatal-specific failure to thrive and loss of macromolecular nutrient uptake. RNA-Seq and CUT&RUN analyses defined an endo-lysosomal program as being downstream of these transcription factors. We demonstrate major transcriptional changes in metabolic pathways, including fatty acid oxidation and increases in peroxisome number, in response to loss of Maf proteins. Finally, we show that loss of BLIMP1, a repressor of adult enterocyte genes, shows highly overlapping changes in gene expression and similar defects in macromolecular uptake. This work defines transcriptional regulators that are necessary for nutrient uptake in neonatal enterocytes.

Genomic analysis of 1,25-dihydroxyvitamin D3 action in mouse intestine reveals compartment and segment-specific gene regulatory effects.

1,25-dihydroxyvitamin D (VD) regulates intestinal calcium absorption in the small intestine (SI) and also reduces risk of colonic inflammation and cancer. However, the intestine compartment-specific target genes of VD signaling are unknown. Here, we examined VD action across three functional compartments of the intestine using RNA-seq to measure VD-induced changes in gene expression and Chromatin Immunoprecipitation with next generation sequencing to measure vitamin D receptor (VDR) genomic binding. We found that VD regulated the expression of 55 shared transcripts in the SI crypt, SI villi, and in the colon, including Cyp24a1, S100g, Trpv6, and Slc30a10. Other VD-regulated transcripts were unique to the SI crypt (162 up, 210 down), villi (199 up, 63 down), or colon (102 up, 28 down), but this did not correlate with mRNA levels of the VDR. Furthermore, bioinformatic analysis identified unique VD-regulated biological functions in each compartment. VDR-binding sites were found in 70% of upregulated genes from the colon and SI villi but were less common in upregulated genes from the SI crypt and among downregulated genes, suggesting some transcript-level VD effects are likely indirect. Consistent with this, we show that VD regulated the expression of other transcription factors and their downstream targets. Finally, we demonstrate that compartment-specific VD-mediated gene expression was associated with compartment-specific VDR-binding sites (<30% of targets) and enrichment of intestinal transcription factor-binding motifs within VDR-binding peaks. Taken together, our data reveal unique spatial patterns of VD action in the intestine and suggest novel mechanisms that could account for compartment-specific functions of this hormone.

Structure-activity relationships of 1,4-bis(arylsulfonamido)-benzene or naphthalene-N,N'-diacetic acids with varying C2-substituents as inhibitors of Keap1-Nrf2 protein-protein interaction.

The Keap1-Nrf2-ARE pathway plays an important role in responding to oxidative stress and maintaining the redox homeostasis. Small molecule inhibitors targeting directly the Keap1-Nrf2 protein-protein interaction (PPI) can potentially be developed into effective preventive and therapeutic agents for numerous chronic inflammatory diseases. To improve the drug-like properties and inhibitory potency of these inhibitors, a series of 1,4-bis(arylsulfonamido)benzene or naphthalene-N,N'-diacetic acids with varying substituents at C-2 position of the benzene or naphthalene core were designed and synthesized. Among them, compound 12d with 2-(4-fluorobenzyloxy) group was the most potent direct inhibitor of Keap1-Nrf2 PPI with an IC50 of 64.5 nM in the fluorescent polarization (FP) assay and 14.2 nM in a time-resolved fluorescence resonance energy transfer (TR-FRET) assay. Moreover, cell-based biological assay showed that 12d significantly increased the mRNA levels of Nrf2 downstream genes, GSTM3, HMOX2 and NQO1, through Nrf2 activation. The discovery of the new scaffolds possessing diverse O-linked fragments at the C2 position offers opportunities to further modify the chemical structures of Keap1-Nrf2 PPI inhibitors to improve their pharmacokinetic, efficacy and safety profiles.

Drivers of transcriptional variance in human intestinal epithelial organoids.

Human intestinal epithelial organoids (enteroids and colonoids) are tissue cultures used for understanding the physiology of the human intestinal epithelium. Here, we explored the effect on the transcriptome of common variations in culture methods, including extracellular matrix substrate, format, tissue segment, differentiation status, and patient heterogeneity. RNA-sequencing datasets from 276 experiments performed on 37 human enteroid and colonoid lines from 29 patients were aggregated from several groups in the Texas Medical Center. DESeq2 and gene set enrichment analysis (GSEA) were used to identify differentially expressed genes and enriched pathways. PERMANOVA, Pearson's correlation, and dendrogram analysis of the data originally indicated three tiers of influence of culture methods on transcriptomic variation: substrate (collagen vs. Matrigel) and format (3-D, transwell, and monolayer) had the largest effect; segment of origin (duodenum, jejunum, ileum, colon) and differentiation status had a moderate effect; and patient heterogeneity and specific experimental manipulations (e.g., pathogen infection) had the smallest effect. GSEA identified hundreds of pathways that varied between culture methods, such as IL1 cytokine signaling enriched in transwell versus monolayer cultures and E2F target genes enriched in collagen versus Matrigel cultures. The transcriptional influence of the format was furthermore validated in a synchronized experiment performed with various format-substrate combinations. Surprisingly, large differences in organoid transcriptome were driven by variations in culture methods such as format, whereas experimental manipulations such as infection had modest effects. These results show that common variations in culture conditions can have large effects on intestinal organoids and should be accounted for when designing experiments and comparing results between laboratories. Our data constitute the largest RNA-seq dataset interrogating human intestinal epithelial organoids.

FILIP1L Loss Is a Driver of Aggressive Mucinous Colorectal Adenocarcinoma and Mediates Cytokinesis Defects through PFDN1.

Aneuploid mucinous colorectal adenocarcinoma (MAC) is an aggressive subtype of colorectal cancer with poor prognosis. The tumorigenic mechanisms in aneuploid MAC are currently unknown. Here we show that downregulation of Filamin A-interacting protein 1-like (FILIP1L) is a driver of MAC. Loss of FILIP1L increased xenograft growth, and, in colon-specific knockout mice, induced colonic epithelial hyperplasia and mucin secretion. The molecular chaperone prefoldin 1 (PFDN1) was identified as a novel binding partner of FILIP1L at the centrosomes throughout mitosis. FILIP1L was required for proper centrosomal localization of PFDN1 and regulated proteasome-dependent degradation of PFDN1. Importantly, increased PFDN1, caused by downregulation of FILIP1L, drove multinucleation and cytokinesis defects in vitro and in vivo, which were confirmed by time-lapse imaging and 3D cultures of normal epithelial cells. Overall, these findings suggest that downregulation of FILIP1L and subsequent upregulation of PFDN1 is a driver of the unique neoplastic characteristics in aggressive aneuploid MAC. SIGNIFICANCE: This study identifies FILIP1L as a tumor suppressor in mucinous colon cancer and demonstrates that FILIP1L loss results in aberrant stabilization of a centrosome-associated chaperone protein to drive aneuploidy and disease progression.

SMAD4 is critical in suppression of BRAF-V600E serrated tumorigenesis.

BRAF-driven colorectal cancer is among the poorest prognosis subtypes of colon cancer. Previous studies suggest that BRAF-mutant serrated cancers frequently exhibit Microsatellite Instability (MSI) and elevated levels of WNT signaling. The loss of tumor-suppressor Smad4 in oncogenic BRAF-V600E mouse models promotes rapid serrated tumor development and progression, and SMAD4 mutations co-occur in human patient tumors with BRAF-V600E mutations. This study assesses the role of SMAD4 in early-stage serrated tumorigenesis. SMAD4 loss promotes microsatellite stable (MSS) serrated tumors in an oncogenic BRAF-V600E context, providing a model for MSS serrated cancers. Inactivation of Msh2 in these mice accelerated tumor formation, and whole-exome sequencing of both MSS and MSI serrated tumors derived from these mouse models revealed that all serrated tumors developed oncogenic WNT mutations, predominantly in the WNT-effector gene Ctnnb1 (ß-catenin). Mouse models mimicking the oncogenic ß-catenin mutation show that the combination of three oncogenic mutations (Ctnnb1, Braf, and Smad4) are critical to drive rapid serrated dysplasia formation. Re-analysis of human tumor data reveals BRAF-V600E mutations co-occur with oncogenic mutations in both WNT and SMAD4/TGFß pathways. These findings identify SMAD4 as a critical factor in early-stage serrated cancers and helps broaden the knowledge of this rare but aggressive subset of colorectal cancer.

Moringa isothiocyanate-1 regulates Nrf2 and NF-κB pathway in response to LPS-driven sepsis and inflammation.

This study aims to document the dual mode of pharmacological action of moringa isothiocyanate-1 (MIC-1) derived from seeds of Moringa oleifera Lam. Oral administration of chemically stable MIC-1 (80 mg/kg) significantly reduced the expression of inflammatory markers (Tnf-α, Ifn-α, IL-1ß, IL-6) in the liver, kidney, spleen, and colon and decreased spleen weight in the lipopolysaccharide (LPS)-induced sepsis / acute inflammation model in mice. Transcriptomic analysis of the effect of MIC-1 on the liver and in the LPS-induced RAW264.7 murine macrophage showed that MIC-1 decreases inflammation via inflammation, immunity, and oxidative stress pathways. These results are supported by the immunocytochemical observations that MIC-1 increased the nuclear accumulation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) transcription factor and decreased the nuclear accumulation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in the LPS-induced macrophages. Transcriptional activation of antioxidant genes by MIC-1 translated into a reduction of reactive oxygen species (ROS) in the cytoplasm, decrease of mitochondrial superoxide content, and restoration of the mitochondrial membrane potential in the LPS-induced macrophages. Our data indicate that MIC-1 affects inflammation and oxidative stress, two key processes involved in the etiology of many chronic diseases. These effects involve upstream regulation of two key transcriptional factors regulating responses to these processes at a gene expression level.

SOX4 and SMARCA4 cooperatively regulate PI3k signaling through transcriptional activation of TGFBR2.

Dysregulation of PI3K/Akt signaling is a dominant feature in basal-like or triple-negative breast cancers (TNBC). However, the mechanisms regulating this pathway are largely unknown in this subset of aggressive tumors. Here we demonstrate that the transcription factor SOX4 is a key regulator of PI3K signaling in TNBC. Genomic and proteomic analyses coupled with mechanistic studies identified TGFBR2 as a direct transcriptional target of SOX4 and demonstrated that TGFBR2 is required to mediate SOX4-dependent PI3K signaling. We further report that SOX4 and the SWI/SNF ATPase SMARCA4, which are uniformly overexpressed in basal-like tumors, form a previously unreported complex that is required to maintain an open chromatin conformation at the TGFBR2 regulatory regions in order to mediate TGFBR2 expression and PI3K signaling. Collectively, our findings delineate the mechanism by which SOX4 and SMARCA4 cooperatively regulate PI3K/Akt signaling and suggest that this complex may play an essential role in TNBC genesis and/or progression.

LIN28B induces a differentiation program through CDX2 in colon cancer.

Most colorectal cancers (CRCs) are moderately differentiated or well differentiated, a status that is preserved even in metastatic tumors. However, the molecular mechanisms underlying CRC differentiation remain to be elucidated. Herein, we unravel a potentially novel posttranscriptional regulatory mechanism via a LIN28B/CDX2 signaling axis that plays a critical role in mediating CRC differentiation. Owing to a large number of mRNA targets, the mRNA-binding protein LIN28B has diverse functions in development, metabolism, tissue regeneration, and tumorigenesis. Our RNA-binding protein IP (RIP) assay revealed that LIN28B directly binds CDX2 mRNA, which is a pivotal homeobox transcription factor in normal intestinal epithelial cell identity and differentiation. Furthermore, LIN28B overexpression resulted in enhanced CDX2 expression to promote differentiation in subcutaneous xenograft tumors generated from CRC cells and metastatic tumor colonization through mesenchymal-epithelial transition in CRC liver metastasis mouse models. A ChIP sequence for CDX2 identified α-methylacyl-CoA racemase (AMACR) as a potentially novel transcriptional target of CDX2 in the context of LIN28B overexpression. We also found that AMACR enhanced intestinal alkaline phosphatase activity, which is known as a key component of intestinal differentiation, through the upregulation of butyric acid. Overall, we demonstrated that LIN28B promotes CRC differentiation through the CDX2/AMACR axis.

Requirement of Bccip for the Regeneration of Intestinal Progenitors.

BCCIP was originally identified as a BRCA2 and CDKN1A/p21 interaction protein. Although a partial loss of BCCIP function is sufficient to trigger genomic instability and tumorigenesis, complete deletion of BCCIP is lethal to cells. Using Rosa26-CreERT2 mouse models, we found that induced Bccip deletion in adult mice caused an acute intestinal epithelial denudation that cannot be relieved by co-deletion of Trp53. The critical role of Bccip in intestine epithelial renewal was verified with a Villin-CreERT2 mouse model. The epithelium degeneration was associated with a rapid loss of the proliferative capability of the crypt progenitor cells in vivo, lack of crypt base columnar stem cell markers, and a failure of in vitro crypt organoid growth. RNA-Seq analysis of freshly isolated intestinal crypt cells showed that Bccip deletion caused an overwhelming down-regulation of genes involved in mitotic cell division but an up-regulation of genes involved in apoptosis and stress response to microbiomes. Our data not only indicate that intestinal epithelium is the most sensitive tissue to whole-body deletion of Bccip but also point to Bccip as a novel and critical factor for the proliferation of the intestinal progenitors. These findings have significant implications for understanding why a hypomorphic loss of BCCIP functions is more relevant to tumorigenesis.

Analysis of 1,25-Dihydroxyvitamin D3 Genomic Action Reveals Calcium-Regulating and Calcium-Independent Effects in Mouse Intestine and Human Enteroids.

Although vitamin D is critical for the function of the intestine, most studies have focused on the duodenum. We show that transgenic expression of the vitamin D receptor (VDR) only in the distal intestine of VDR null mice (KO/TG mice) results in the normalization of serum calcium and rescue of rickets. Although it had been suggested that calcium transport in the distal intestine involves a paracellular process, we found that the 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]-activated genes in the proximal intestine associated with active calcium transport (Trpv6, S100g, and Atp2b1) are also induced by 1,25(OH)2D3 in the distal intestine of KO/TG mice. In addition, Slc30a10, encoding a manganese efflux transporter, was one of the genes most induced by 1,25(OH)2D3 in both proximal and distal intestine. Both villus and crypt were found to express Vdr and VDR target genes. RNA sequence (RNA-seq) analysis of human enteroids indicated that the effects of 1,25(OH)2D3 observed in mice are conserved in humans. Using Slc30a10-/- mice, a loss of cortical bone and a marked decrease in S100g and Trpv6 in the intestine was observed. Our findings suggest an interrelationship between vitamin D and intestinal Mn efflux and indicate the importance of distal intestinal segments to vitamin D action.

Paneth Cell-Derived Lysozyme Defines the Composition of Mucolytic Microbiota and the Inflammatory Tone of the Intestine.

Paneth cells are the primary source of C-type lysozyme, a ß-1,4-N-acetylmuramoylhydrolase that enzymatically processes bacterial cell walls. Paneth cells are normally present in human cecum and ascending colon, but are rarely found in descending colon and rectum; Paneth cell metaplasia in this region and aberrant lysozyme production are hallmarks of inflammatory bowel disease (IBD) pathology. Here, we examined the impact of aberrant lysozyme production in colonic inflammation. Targeted disruption of Paneth cell lysozyme (Lyz1) protected mice from experimental colitis. Lyz1-deficiency diminished intestinal immune responses to bacterial molecular patterns and resulted in the expansion of lysozyme-sensitive mucolytic bacteria, including Ruminococcus gnavus, a Crohn's disease-associated pathobiont. Ectopic lysozyme production in colonic epithelium suppressed lysozyme-sensitive bacteria and exacerbated colitis. Transfer of R. gnavus into Lyz1-/- hosts elicited a type 2 immune response, causing epithelial reprograming and enhanced anti-colitogenic capacity. In contrast, in lysozyme-intact hosts, processed R. gnavus drove pro-inflammatory responses. Thus, Paneth cell lysozyme balances intestinal anti- and pro-inflammatory responses, with implications for IBD.