Abstract

BACKGROUND AND AIMS:

During active inflammation tissue intraluminal intestinal pH is decreased in patients with inflammatory bowel disease (IBD). Acidic pH may play a role in IBD pathophysiology. Recently, proton sensing G-protein coupled receptors were identified, including GPR4, OGR1 (GPR68), and TDAG8 (GPR65). We investigated whether GPR4 is involved in intestinal inflammation.

METHODS:

The role of GPR4 was assessed in murine colitis models: chronic dextran sulphate sodium (DSS) administration and by crossbreeding into an IL-10 deficient background for development of spontaneous colitis. Colitis severity was assessed by body weight, colonoscopy, colon length, histological score, cytokine mRNA expression, and myeloperoxidase (MPO) activity. In the spontaneous Il-10-/- colitis model, the incidence of rectal prolapse and characteristics of lamina propria leukocytes (LPLs) were analyzed.

RESULTS:

Gpr4-/- mice showed reduced body weight loss and histology score after induction of chronic DSS colitis. In Gpr4-/- /Il-10-/- double knock-outs the onset and progression of rectal prolapse were significantly delayed and mitigated compared to Gpr4+/+ /Il-10-/- mice. Double knock-out mice showed lower histology scores, MPO activity, CD4 + T-helper cell infiltration, IFN-γ, iNOS, MCP-1 (CCL2), CXCL1 and CXCL2 expression compared to controls. In colon, GPR4 mRNA was detected in endothelial cells, some smooth muscle cells, and some macrophages.

CONCLUSION:

Absence of GPR4 ameliorates colitis in IBD animal models indicating an important regulatory rolein mucosal inflammation, thus providing a new link between tissue pH and the immune system. Therapeutic inhibition of GPR4 may be beneficial for the treatment of IBD.

Loss of intracellular calcium homeostasis may contribute to the opacification of lens tissue during cortical cataract formation. In healthy lenses, the concentration of intracellular calcium is maintained at levels far below electrochemical equilibrium but the identity of the calcium extrusion mechanism in lens fiber cells has remained elusive. Previous studies focused on the role of plasma membrane calcium ATPases and sodium-calcium exchangers. Here, we examined the expression of mRNA transcripts encoding potassium-dependent sodium-calcium exchangers (Nckx's, encoded by the Slc24 gene family) in the mouse lens. The most abundant of the five Slc24 family members was Slc24a4 (Nckx4). Notably, Slc24a4 was the only family member with increased expression in fiber cells. Using an antibody raised against recombinant mouse Nckx4, we showed that the protein is expressed strongly in the outer cortical fibers, consistent with results of in situ hybridizationexperiments and earlier mass spectrometry analysis. To test the role of Nckx4 directly, we generated mice in which Slc24a4 was deleted conditionally in lens tissue. In conditional knockout animals, the level of Nckx4 protein was reduced to background levels without a discernible effect on lens growth or transparency. Thus, despite its relative abundance in the lens, Nckx4 does not appear to have an indispensable role in the maintenance of lens clarity.

The gene PTCHD1 is mutated in patients with autism spectrum disorders (ASD) and intellectual disabilities (ID) and has been hypothesized to contribute to Sonic hedgehog (Shh) signaling and synapse formation. We identify a panel of Ptchd1 interacting proteins that include postsynaptic density proteins and the retromer complex, revealing a link to critical regulators of dendritic and postsynaptic trafficking. Ptchd1knock-out male mice exhibit cognitive alterations, including defects in a novel object recognition task. To test whether Ptchd1 is required for Shh-dependent signaling, we examined two Shh-dependent cell populations that express high levels of Ptchd1 mRNA: cerebellar granule cell precursors and dentate granule cells in the hippocampus. We find that proliferation of these neuronal precursors is not significantly altered in Ptchd1 knock-out male mice. We used whole-cell electrophysiology and anatomical methods to assess synaptic function in Ptchd1-deficient dentate granule cells. In the absence of Ptchd1, we observed profound disruption in excitatory/inhibitory balance despite normal dendritic spine density on dentate granule cells. These findings support a critical role of Ptchd1 protein in the dentate gyrus but indicate that it is not required for structural synapse formation in dentate granule cells or for Shh-dependent neuronal precursor proliferation.

SIGNIFICANCE STATEMENT
The mechanisms underlying neuronal and cellular alterations resulting from Ptchd1 mutations are unknown. The results from this study support an association with dendritic trafficking complexes of Ptchd1. Loss-of-function experiments do not support a role in sonic-hedgehog-dependent signaling but reveal a disruption of synaptic transmission in the mouse dentate gyrus. The findings will help guide ongoing efforts on understanding the etiology of neurodevelopmental disorders arising from Ptchd1-deficiency.

Endothelial cells (ECs) are a key component of the tumor microenvironment. They have abnormal characteristics compared to the ECs in normal tissues. Here, we found a marked increase in lipocalin-type prostaglandin D synthase (L-PGDS) mRNA (Ptgds) expression in ECs isolated from mouse melanoma. Immunostaining of mouse melanoma revealed expression of L-PGDS protein in the ECs. In situhybridization also showed L-PGDS (PTGDS) mRNA expression in the ECs of human melanoma and oral squamous cell carcinoma. In vitro experiments showed that stimulation with tumor cell-derived IL-1 and TNF-α increased L-PGDS mRNA expression and its product prostaglandin D2 (PGD2 ) in human normal ECs. We also investigated the contribution of L-PGDS-PGD2 to tumor growth and vascularization. Systemic or EC-specific deficiency of L-PGDS accelerated the growth of melanoma in mice, whereas treatment with an agonist of the PGD2receptor, DP1 (BW245C, 0.1 mg/kg, injected intraperitoneally twice daily), attenuated it. Morphological and in vivo studies showed that endothelial L-PGDS deficiency resulted in functional changes of tumor ECs such as accelerated vascular hyperpermeability, angiogenesis, and endothelial-to-mesenchymal transition (EndMT) in tumors, which in turn reduced tumor cell apoptosis. These observations suggest that tumor cell-derived inflammatory cytokines increase L-PGDS expression and subsequent PGD2 production in the tumor ECs. This PGD2 acts as a negative regulator of the tumorigenic changes in tumor ECs.

Claudin-2 is a trans-membrane protein-component of tight junctions in epithelial cells. Elevated claudin-2 expression has been reported in colorectal cancer (CRC). The aim of this study was to investigate the expression patterns of claudin-2 in human CRC samples and analyze its association with clinical characteristics and treatment outcome. TMAs of primary tumors from two cohorts of metastatic CRC (mCRC) were used. Claudin-2 IHC staining was evaluated in a semi-quantitative manner in different regions and cell types. Claudin-2 expression was also analyzed by immunofluorescence in primary cultures of human CRC cancer-associated fibroblasts (CAFs). Initial analyses identified previously unrecognized expression patterns of claudin-2 in CAFs of human CRC. Claudin-2 expression in CAFs of the invasive margin was associated with shorter progression-free survival. Subgroup analyses demonstrated that the survival associations occurred among cases that received 5-FU+oxaliplatin combination treatment, but not in patients receiving 5-FU±irinotecan. The finding was validated by analyses of the independent cohort. In summary, previously unreported stromal expression of claudin-2 in CAFs of human CRC was detected together with significant association between high claudin-2 expression in CAFs and shorter survival in 5-FU+oxaliplatin-treated mCRC patients.

The cerebellum undergoes rapid growth during the third trimester and is vulnerable to injury and deficient growth in infants born prematurely. Factors associated with preterm cerebellar hypoplasia include chronic lung disease and postnatal glucocorticoid administration. We modeled chronic hypoxemia and glucocorticoid administration in neonatal mice to study whole cerebellar and cell type-specific effects of dual exposure. Chronic neonatal hypoxia resulted in permanent cerebellar hypoplasia. This was compounded by administration of prednisolone as shown by greater volume loss and Purkinje cell death. In the setting of hypoxia and prednisolone, administration of a small molecule Smoothened-Hedgehog agonist (SAG) preserved cerebellar volume and protected against Purkinje cell death. Such protective effects were observed even when SAG was given as a one-time dose after dual insult. To model complex injury and determine cell type-specific roles for the hypoxia inducible factor (HIF) pathway, we performed conditional knockout of von Hippel Lindau (VHL) to hyperactivate HIF1α in cerebellar granule neuron precursors (CGNP) or Purkinje cells. Surprisingly, HIF activation in either cell type resulted in no cerebellar deficit. However, in mice administered prednisolone, HIF overactivation in CGNPs resulted in significant cerebellar hypoplasia, whereas HIF overactivation in Purkinje cells caused cell death. Together, these findings indicate that HIF primes both cell types for injury via glucocorticoids, and that hypoxia/HIF + postnatal glucocorticoid administration act on distinct cellular pathways to cause cerebellar injury. They further suggest that SAG is neuroprotective in the setting of complex neonatal cerebellar injury.

Brain regions that regulate fluid satiation are not well characterized, yet are essential for understanding fluid homeostasis. We found that oxytocin-receptor-expressing neurons in the parabrachial nucleus of mice (OxtrPBN neurons) are key regulators of fluid satiation. Chemogenetic activation of OxtrPBN neurons robustly suppressed noncaloric fluid intake, but did not decrease food intake after fasting or salt intake following salt depletion; inactivation increased saline intake after dehydration and hypertonic saline injection. Under physiological conditions, OxtrPBN neurons were activated by fluid satiation and hypertonic saline injection. OxtrPBN neurons were directly innervated by oxytocin neurons in the paraventricular hypothalamus (OxtPVH neurons), which mildly attenuated fluid intake. Activation of neurons in the nucleus of the solitary tract substantially suppressed fluid intake and activated OxtrPBN neurons. Our results suggest that OxtrPBN neurons act as a key node in the fluid satiation neurocircuitry, which acts to decrease water and/or saline intake to prevent or attenuate hypervolemia and hypernatremia.

The chicken yolk sac (YS) and small intestine are essential for nutrient absorption during the pre-hatch and post-hatch periods, respectively. Absorptive enterocytes and secretory cells line the intestinal villi and originate from stem cells located in the intestinal crypts. Similarly, in the YS, there are absorptive and secretory cells that presumably originate from a stem cell population. Leucine-rich repeat containing G protein-coupled receptor 5 (Lgr5) and olfactomedin 4 (Olfm4) are 2 widely used markers for intestinal stem cells. The objective of this study was to map the distribution of putative stem cells expressing LGR5 and OLFM4 mRNA in the chicken small intestine from the late embryonic period to early post hatch and the YS during embryogenesis. At embryonic d 11, 13, 15, 17, and 19, the YS was collected (n = 3), and small intestine was collected at embryonic d 19, d of hatch (doh), and d 1, 4, and 7 post hatch (n = 3). Cells expressing OLFM4 and LGR5 mRNA were identified by in situ hybridization. In the YS, cells expressing only LGR5 and not OLFM4 mRNA were localized to the vascular endothelial cells lining the blood vessels. In the small intestine, cells in the intestinal crypt expressed both LGR5 and OLFM4 mRNA. Staining for OLFM4 mRNA was more intense than LGR5 mRNA, demonstrating that Olfm4 is a more robust marker for stem cells than Lgr5. At embryonic d 19 and doh, cells staining for OLFM4 mRNA were already present in the rudimentary crypts, with the greatest staining in the duodenal crypts. The intensity of OLFM4 mRNA staining increased from doh to d 7 post hatch. Dual label staining at doh for the peptide transporter PepT1 and Olfm4 revealed a population of cells above the crypts that did not express Olfm4 or PepT1 mRNA. These cells are likely progenitor transit amplifying cells. Thus, avians and mammals share similarity in the ontogeny of stem cells in the intestinal crypts.

Pericytes are mural cells of blood microvessels which play a crucial role at the neurovascular interface of the central nervous system. They are involved in the regulation of blood-brain barrier integrity, angiogenesis, clearance of toxic metabolites, capillary hemodynamic responses, and neuroinflammation, and they demonstrate stem cell activity. Morphological and molecular studies to characterize brain pericytes recently pointed out some heterogeneity in pericyte population. Nevertheless, a clear definition of pericyte subtypes is still lacking. Here, we demonstrate that a fraction of brain pericytes express Connexin 30 (Cx30), a gap junction protein, which, in the brain parenchyma, was thought to be exclusively found in astrocytes. Cx30 could thus be a candidate protein in the composition of the gap junction channels already described between endothelial cells and pericytes. It could also form hemichannels or acts in a channel-independent manner to regulate pericyte morphology, as already observed in astrocytes. Altogether, our results suggest that Cx30 defines a novel brain pericyte subtype.

Bovine γδ T cells are amongst the first cells to accumulate at the site of Mycobacterium bovis infection; however, their role in the developing lesion remains unclear. We utilized transcriptomics analysis, in situ hybridization, and a macrophage/γδ T cell co-culture system to elucidate the role of γδ T cells in local immunity to M. bovis infection. Transcriptomics analysis revealed that γδ T cells upregulated expression of several novel, immune-associated genes in response to stimulation with M. bovis antigen. BCG-infected macrophage/γδ T cell co-cultures confirmed the results of our RNAseq analysis, and revealed that γδ T cells from M. bovis-infected animals had a significant impact on bacterial viability. Analysis of γδ T cells within late-stage M. bovis granulomas revealed significant expression of IFN-γ and CCL2, but not IL-10, IL-22, or IL-17. Our results suggest γδ T cells influence local immunity to M. bovis through cytokine secretion and direct effects on bacterial burden.

Intestinal epithelial cells absorb nutrients, respond to microbes, function as a barrier and help to coordinate immune responses. Here we report profiling of 53,193 individual epithelial cells from the small intestine and organoids of mice, which enabled the identification and characterization of previously unknown subtypes of intestinal epithelial cell and their gene signatures. We found unexpected diversity in hormone-secreting enteroendocrine cells and constructed the taxonomy of newly identified subtypes, and distinguished between two subtypes of tuft cell, one of which expresses the epithelial cytokine Tslp and the pan-immune marker CD45, which was not previously associated with non-haematopoietic cells. We also characterized the ways in which cell-intrinsic states and the proportions of different cell types respond to bacterial and helminth infections: Salmonella infection caused an increase in the abundance of Paneth cells and enterocytes, and broad activation of an antimicrobial program; Heligmosomoides polygyrus caused an increase in the abundance of goblet and tuft cells. Our survey highlights previously unidentified markers and programs, associates sensory molecules with cell types, and uncovers principles of gut homeostasis and response to pathogens.

Activating mutations in the Wnt pathway are a characteristic feature of colorectal cancer (CRC). The R-spondin (RSPO) family is a group of secreted proteins that enhance Wnt signaling and RSPO2 and RSPO3 gene fusions have been reported in CRC. We have previously shown that Wnt pathway blockers exhibit potent combinatorial activity with taxanes to inhibit tumor growth. Here we show that RSPO3 antagonism synergizes with paclitaxel based chemotherapies in patient-derived xenograft models (PDX) with RSPO3 fusions and in tumors with common CRC mutations such as APC, β-catenin, or RNF43. In these latter types of tumors that represent over 90% of CRC, RSPO3 is produced by stromal cells in the tumor microenvironment and the activating mutations appear to sensitize the tumors to Wnt-Rspo synergy. The combination of RSPO3 inhibition and taxane treatment provides an approach to effectively target oncogenic WNT signaling in a significant number of patients with colorectal and other intestinal cancers.