Jennifer Hawkins’s research while affiliated with Cincinnati Children's Hospital Medical Center and other places

Sonic Hedgehog (Shh), secreted from gastric parietal cells, contributes to the regeneration of the epithelium. The recruitment of macrophages plays a central role in the regenerative process. The mechanism that regulates macrophage recruitment in response to gastric injury is largely unknown. Here we tested the hypothesis that Shh stimulates macrophage chemotaxis to the injured epithelium and contributes to gastric regeneration. A mouse model expressing a myeloid cell-specific deletion of Smoothened (LysMcre/+;Smof/f) was generated using transgenic mice bearing loxP sites flanking the Smo gene (Smo loxP) and mice expressing a Cre recombinase transgene from the Lysozyme M locus (LysMCre). Acetic acid injury was induced in the stomachs of both control and LysMcre/+;Smof/f (SmoKO) mice and gastric epithelial regeneration and macrophage recruitment analyzed over a period of 7 days post-injury. Bone marrow-derived macrophages (BM-Mø) were collected from control and SmoKO mice. Human-derived gastric organoid/macrophage co-cultures were established, and macrophage chemotaxis measured. Compared to control mice, SmoKO animals exhibited inhibition of ulcer repair and normal epithelial regeneration, which correlated with decreased macrophage infiltration at the site of injury. Bone marrow chimera experiments using SmoKO donor cells showed that control chimera mice transplanted with SmoKO bone marrow donor cells exhibited a loss of ulcer repair, and transplantation of control bone marrow donor cells to SmoKO mice rescued epithelial cell regeneration. Histamine-stimulated Shh secretion in human organoid/macrophage co-cultures resulted in macrophage migration toward the gastric epithelium, a response that was blocked with Smo inhibitor Vismodegib. Shh-induced macrophage migration was mediated by AKT signaling. In conclusion, Shh signaling acts as a macrophage chemoattractant via a Smo-dependent mechanism during gastric epithelial regeneration in response to injury.

(1) Background: The expression of programmed death-ligand 1 (PD-L1), which interacts with programmed cell death protein 1 (PD-1) on cytotoxic T lymphocytes (CTLs), enables tumors to escape immunosurveillance. The PD-1/PD-L1 interaction results in the inhibition of CTL proliferation, and effector function, thus promoting tumor cell evasion from immunosurveillance and cancer persistence. Despite 40% of gastric cancer patients exhibiting PD-L1 expression, only a small subset of patients responds to immunotherapy. Human epidermal growth factor receptor2 (HER2) is one of the critical regulators of several solid tumors, including metastatic gastric cancer. Although half of PD-L1-positive gastric tumors co-express HER2, crosstalk between HER2 and PD-1/PD-L1 in gastric cancer remains undetermined. (2) Methods: Human gastric cancer organoids (huTGOs) were generated from biopsied or resected tissues and co-cultured with CTLs and myeloid-derived suppressor cells (MDSCs). Digital Spatial Profiling (DSP) was performed on FFPE tissue microarrays of numerous gastric cancer patients to examine the protein expression of immune markers. (3) Results: Knockdown of HER2 in PD-L1/HER2-positive huTGOs led to a concomitant decrease in PD-L1 expression. Similarly, in huTGOs/immune cell co-cultures, PD-L1 expression decreased in huTGOs and was correlated with an increase in CTL proliferation which enhanced huTGO death. Treatment with Nivolumab exhibited similar effects. However, a combinatorial treatment with Mubritinib and Nivolumab was unable to inhibit HER2 expression in co-cultures containing MDSCs. (4) Conclusions: Our study suggested that co-expression of HER2 and PD-L1 may contribute to tumor cell immune evasion. In addition, autologous organoid/immune cell co-cultures can be exploited to effectively screen responses to a combination of anti-HER2 and immunotherapy to tailor treatment for gastric cancer patients.

Background Increased incidence of gastric ulcer disease is often related to frequent nonsteroidal anti‐inflammatory drug (NSAID) use and Helicobacter pylori ( H. pylori ) infection. Studies have shown H. pylori adheres to the gastric epithelium through binding of bacterial Sialic Acid‐binding Adhesin (SAbA) to glycosylated gastric epithelial mucin receptor sialyl‐Lewis X (S‐Lew X ). Our group has reported that CD44v9 emerges during repair of the gastric epithelium after injury where it is co‐expressed with markers of Spasmolytic Polypeptide/TFF2‐Expressing Metaplasia (SPEM), a reparative phenotype which has been shown to emerge in response to gastric injury. H. pylori localizes specifically to SPEM glands within the corpus of the stomach. We hypothesize that SAbA adhesin and S‐Lew X facilitate H. pylori preferential localization and adhesion to SPEM glands in response to injury. Hypothesis The aged gastric epithelium exhibits sustained SPEM glands expressing S‐Lew X in response to injury. H. pylori preferential localization and adhesion to SPEM glands may contribute to the severity of disease in the aged epithelium. Methods Gastric ulcers were induced in young (2–3 months old) and aged (>8 months old) C57/BL6 mice using an acetic acid‐induced injury model. Twenty‐one days after ulcer injury, mice were inoculated with H. pylori or with Brucella broth. Gastric organoids were generated from biopsies collected from young (14–30 years) or elderly (>55 years) human patients and transferred to monolayer cultures for in vivo H. pylori adherence assays. Infected monolayers were treated with sialic acid analogs 3′‐sialyllactose or 6′‐sialyllactose. Flow cytometry and immunofluorescence was used to analyze the co‐localization and adherence of H. pylori to S‐Lew X ‐expressing SPEM glands. Results Sixty days post‐injury, MUC5AC re‐localized to the surface of the gastric epithelium with complete regeneration of the gastric epithelium, expression of parietal, chief and endocrine cells and no evidence of SPEM in the young mice. The aged gastric epithelium exhibited decreased and irregular MUC5AC localization and the persistence of SPEM glands that expressed both CD44v9 and S‐Lew X . Twenty‐one days post‐injury, H. pylori adherence was limited to the surface epithelium in young mice. However, in the aged mice, metaplasia was present, chief cells were decreased, and H. pylori adherence was localized at the base of the epithelial glands and co‐localized with CD44v9/S‐Lew X ‐expressing glands. Adherence assays demonstrated that H. pylori adherence was preferential to □‐2,3‐sialyl‐glycans. Conclusion Following injury in aged gastric tissues, the mucous layer becomes altered such that SPEM persists and there is loss of normal mucin expression and gastric cell lineages. H. pylori adherence to CD44v9‐positive cells is enhanced by S‐Lew X and abnormal regeneration appears to influence the severity of H. pylori ‐associated disease. Support or Funding Information NIH (NIDDK) 5R01DK083402‐08 grant (YZ); Albert C. Yates Fellowship, Univ. of Cincinnati (MD)

Background & aims: Shiga toxin (Stx) producing Escherichia coli (e.g. O157:H7) infection produces bloody diarrhea, while Stx inhibits protein synthesis and causes the life-threatening systemic complication, hemolytic uremic syndrome (HUS). The murine intestinal tract is resistant to O157:H7 and Stx, and human cells in culture fail to model the complex tissue responses to intestinal injury. We used genetically identical, human stem-cell derived intestinal tissues of varying complexity to study Stx toxicity in vitro and in vivo. Methods: In vitro susceptibility to apical or basolateral exposure to Stx was assessed using human intestinal organoids (HIOs) derived from embryonic stem-cells, or enteroids derived from multipotent intestinal stem cells. HIOs contain a lumen, with a single layer of differentiated epithelium surrounded by mesenchymal cells. Enteroids only contain epithelium. In vivo susceptibility was assessed using HIOs, with or without an enteric nervous system (ENS), transplanted into mice. Results: Stx induced necrosis and apoptotic death in both epithelial and mesenchymal cells. Responses that require protein synthesis (cellular proliferation and wound repair) were also observed. Epithelial barrier function was maintained even after epithelial cell death was seen, and apical to basolateral translocation of Stx was seen. Tissue cross-talk, where mesenchymal cell damage caused epithelial cell damage, was observed. Stx induced mesenchymal expression of the epithelial marker, E-cadherin, the initial step in mesenchymal-epithelial transition. In vivo responses of HIO transplants injected with Stx mirrored those seen in vitro. Conclusions: Intestinal tissue responses to protein synthesis inhibition by Stx are complex. Organoid models allow for an unprecedented examination of human tissue responses to a deadly toxin.

Background/Hypothesis Prevalence of gastric ulcers increase in the elderly, yet the cause of the age‐associated escalation is unknown. Once Helicobacter pylori (H. pylori) enters the stomach lumen, bacterial urease not only converts water & urea to ammonia & bicarbonate to neutralize the acidic environment of the stomach, but also breaks down the protective mucous barrier by breaking down mucin. Subsequently, chronic inflammation induced by H. pylori infection results in epithelial cell death and damage to the stomach lining, also known as a gastric ulcer. We tested the hypothesis that the regenerative process of the aged gastric epithelium is impaired in response to injury, such that there is mis‐localization of mucin expression leading to a compromised protective barrier. Methods Gastric ulcers were induced in young (2–3 months old) and aged (>8 months old) C57/BL6 mice with acetic acid applied on the serosal side of the stomach. Samples were harvested from euthanized mice with ulcer durations from 3 to 60 days. Biopsies were collected from human gastric ulcer patients. Organoids were grown from young mouse gastric epithelium and transplanted at the site of acetic acid‐induced injury in aged mice. Immunofluorescence staining was used to analyze the expression of MUC5AC, MUC2, Griffonia simplicifolia II (GSII), Ulex europeus I (UEAI) , H+, K+‐ATPase, intrinsic factor and chromogranin A. Results Within 3 days post‐injury, there was the emergence of spasmolytic polypeptide‐expressing metaplasia (SPEM) at the ulcer margins of both young and aged gastric epithelium. Sixty days post‐injury there was complete regeneration of the gastric epithelium that exhibited the expression of parietal, chief and endocrine cells, and no evidence of SPEM in the young mice. However, the aged gastric epithelium exhibited the persistence of SPEM glands, expression of MUC2 and gastric cancer stem cell marker CD44v9. Aged mice transplanted with gastric organoids derived from young mice exhibited normal MUC5AC, GSII and UEAI expression similar to young mice 60 days post‐injury. Organoid transplantation of the aged gastric epithelium resulted in the normal expression of gastric cell lineages with an intact mucosal layer. Conclusions Following injury in aged gastric tissues, the mucous layer becomes altered such that SPEM persists uncontrolled and there is loss of normal MUC5AC expression and gastric cell lineages. Ongoing experiments include studies of increased susceptibility to H. pylori ‐induced metaplasia and cancer development using an in vivo acetic acid‐induced injury mouse model and human‐ and mouse‐derived gastric organoids. Support or Funding Information NIH (NIDDK) 5R01DK083402‐08 grant (YZ) This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Background/Hypothesis Hedgehog (Hh) signaling not only plays a crucial role in embryonic development, but also controls gastric physiological events including epithelial cell differentiation, regeneration and acid secretion. Sonic Hedgehog (Shh), is secreted from gastric parietal cells in response to injury and contributes to the regeneration of the injured epithelium. Following injury, the recruitment of macrophages plays crucial role both in the regenerative process. The mechanism that regulates macrophage recruitment in response to injury is largely unknown. Thus, here we tested the hypothesis thatShh stimulates macrophage chemotaxis to the injured epithelium and contributes to gastric regeneration. Methods A mouse model expressing a myeloid cell‐specific deletion of Smoothened (LysMCre/SmoKO) was generated using transgenic mice bearing loxP sites flanking exon 1 of the Smo gene (Smo loxP) and mice expressing a Cre recombinase transgene from the Lysozyme M locus (LysMCre). Acetic acid injury was induced in the stomachs of both control and KO mice and gastric epithelial regeneration and macrophage recruitment analyzed over a period of 7 days post‐injury. Bone marrow‐derived macrophages (BM‐Mø) were collected from control and KO mice. Migration of BM‐Mø was performed using ibidi μ‐Slide Chemotaxis chambers in response to recombinant murine Shh (rmShh). Murine‐ and human‐derived gastric organoid/macrophage co‐cultures were established and macrophage chemotaxis measured. Results 1) LysMCre/SmoKO mice exhibit loss of macrophage recruitment and epithelial regeneration in response to injury: Compared to control mice, KO animals exhibited loss of ulcer repair and normal epithelial regeneration, that correlated with decreased macrophage infiltration at the site of injury. 2) In vitro , macrophages from LysMCre/SmoKO mice or organoid/macrophage co‐cultures treated with Vismodegib lose chemotaxis : Forward migration index, a measure of directed movement, was significantly elevated in control BM‐Mø migrating towards rmShh compared to media control. In contrast, Smo‐deficient BM‐Mø did not migrate towards rmShh. While control BM‐Mø migrated toward chemokine CCL2, this chemotactic response was not observed using Smo‐deficient BM‐Mø. In murine‐derived gastric organoid/BM‐Mø co‐cultures, epithelial Shh, stimulated with histamine, correlated with macrophage migration toward the organoid. This response was lost in organoid/BM‐Mø co‐cultures derived from KO mice. Human‐derived organoid/macrophage co‐cultures stimulated with histamine resulted in Shh secretion and macrophage migration toward the gastric organoid, a response that was blocked with Smo‐inhibitor Vismodegib. Conclusions Shh signaling acts as a macrophage chemoattractant via a Smo‐dependent mechanism during gastric epithelial regeneration in response to injury. Support or Funding Information NIH (NIDDK) 5R01DK083402‐08 grant (YZ) This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Background/Hypothesis Several gastrointestinal functions have been demonstrated to be under circadian clock control and colorectal cancer tissues have been found to have a disruption in core circadian clock components BMAL1 and PER2. Those suffering from chronic Helicobacter pylori ( H . pylori) infection are at a heightened risk of developing gastric adenocarcinoma. Compared to young individuals, the aged population suffer from an increased susceptibility to H. pylori infection as well as diminished expression of circadian clock components which could further predispose them to the development of gastric cancer. We therefore hypothesize that chronic H. pylori infection induces a hyperproliferative response in the gastric epithelium via BMAL1 activation resulting in a susceptibility to gastric cancer. Methods Gastric organoids (hFGOs) derived from primary gastric tissue of either aged (> 55 years of age) or young (14 – 20 years) individuals were generated and subsequently infected with H. pylori . Analysis for proliferation and expression of circadian clock components BMAL1 and PER2 was performed for a period of 24 hours post‐infection. Organoids infected with H. pylori were analyzed for gastric cancer markers by qRT‐PCR. Results Gastric fundic tissue obtained from individuals infected with H. pylori were found to have a significantly upregulated expression of cytoplasmic BMAL1. hFGOs exhibited a robust circadian expression of Bmal1, Per2 , and phosphorylated (inactive) GSK‐3β. In comparison to uninfected controls, hFGOs infected with H. pylori displayed a heightened expression of BMAL1 , phosphorylated GSK‐3β, and epithelial proliferation. Gastric organoids derived from of elderly patients was found to have a diminished rhythmic expression of clock genes Per2 and Bmal1 . This dampening in expression of circadian clock components correlates with an increased expression of markers for intestinal metaplasia. Conclusions Persistent infection by H. pylori inactivates GSK‐3β resulting in the activation of the BMAL1:CLOCK complex leading to a hyperproliferation in gastric epithelial cells. This mechanism could heighten the risk of developing gastric cancer in the aged population and in individuals with chronic H. pylori infection. Per2 knockout mice have been generated and infected with H. pylori in order to further understand this mechanism in vivo . Results are ongoing and pending. Support or Funding Information NIH 1U19AI116491‐01 This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Background/Hypothesis Cluster‐of‐differentiation gene 44 (CD44) is an alternatively spliced cell surface protein. CD44 variant isoform 9 (CD44v9) has been shown to mediate gastric repair, but the precise mechanism is unknown. Reactive Oxygen Species (ROS) driven p38/MAPK signaling leads to increased caveolin‐1, and re‐direction of β‐catenin from the nucleus to the membrane where it decreases fundamental events contributing to repair such as cellular proliferation and migration. CD44v9 stabilizes the cystine‐glutamate transporter, xCT, which provides defense against ROS. Thus, we hypothesized that CD44v9 stabilizes the xCT transporter to potentiate defense against ROS to establish an environment conducive to efficient wound repair. Methods Acetic acid injury was induced in C57BL/6 (BL6) mice followed by vehicle, sulfasalazine (SSZ, an xCT inhibitor) or glutathione inhibitor (GSHinh). Acetic acid gastric injuries were induced in aged mice (>8 months) and animals were treated with p38 inhibitor. Mice were analyzed between days 1 to 30 post‐injury. A novel scratch‐wound assay generated from organoids derived from human stomachs and transferred to a gastric epithelial cell monolayer (huGEM) was treated with vehicle, SSZ or GSHinh. Results 1) CD44v9 and xCT co‐expressing cells emerge at the ulcer margin: CD44v9/xCT co‐expressing cells emerged at the ulcer margin within 3‐days post‐injury. Compared to control mice, animals treated with SSZ or GSHinh showed impaired wound healing and loss of normal epithelial regeneration in response to injury. Western blots showed loss of activated β‐catenin, increased expression of caveolin‐1 that coincided with loss in repair in SSZ‐treated mice. FACS sorted CD44v9+ve cells from the ulcer margin expressed markers of EMT. 2) SSZ and GSHinh inhibited wound healing in huGEM scratch wound assays: HuGEM cultures exhibited a cellular composition reflective of primary gastric tissue that included the expression of parietal, chief, and mucous neck and pit cells. CD44v9/xCT co‐expressing cells emerged at the wound edge in huGEM cultures in response to a scratch wound. HuGEM cultures treated with SSZ or GSHinh exhibited significantly delayed wound closure. 3) p38 inhibitor rescues wound healing in vivo in the stomachs of aged mice: In human gastric biopsies, CD44v9, xCT, and activated β‐catenin was significantly decreased in elderly patients. Aged mice treated with p38 inhibitor displayed increased cellular proliferation, CD44v9 expression, and rescued repair when compared to aged vehicle controls. Conclusion CD44v9 and xCT emerge during gastric regeneration which may potentiate defense against ROS to allow for effective healing. Support or Funding Information R01 DK083402‐06A1 This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Cluster‐of‐differentiation gene 44, in particular CD44 variant isoforms (CD44v), emerges during regeneration of the gastric epithelium in response to injury. In particular, CD44v9 is expressed within Spasmolytic Polypeptide/TFF2‐Expressing Metaplasia (SPEM) glands during gastric repair, but the function is unknown. Here we tested the hypothesis that CD44v9 marks a regenerative cell lineage that plays a functional role in gastric repair. Acetic acid injury was induced in CD44‐deficient (CD44KO) and C57BL/6 (BL6) mice. Mouse‐derived gastric organoids expressing CD44v9 were transplanted at the ulcer site of CD44KO mice. Ulcers, expression of CD44v9, proliferation and histology were measured at days 1, 3, 5 and 7 post‐injury. Human‐derived gastric organoids from elderly patients (>55 years) or young patients (14–20 years) were generated and transplanted into the stomachs of NOD scid gamma (NSG) mice post‐injury. Ulcers were induced in NRGS mice expressing human‐derived immune cells (huNRGS) and the immune phenotype analyzed by CyTOF. CD44v9 expression emerged at the ulcer margin during gastric ulcer repair. Compared to BL6 mice that healed within 7 days post‐injury, CD44KO mice exhibited loss of ulcer repair and gastric epithelial regeneration. Transplantation of CD44v9‐expressing gastric organoids into the stomachs of CD44KO mice promoted ulcer repair. Compared to NSG mice exhibiting loss of CD44v9 expression and gastric repair in response to injury, transplantation of human‐derived gastric organoids from young stomachs promoted repair. Transplantation of organoids derived from aged stomachs did not promote repair. Compared to NRGS mice, huNRGS animals exhibited smaller ulcer sizes and an infiltration of human CD163+ macrophages that correlated with an emergence of CD44v9 expression. Thus, CD44v9 marks a regenerative cell lineage. Macrophages infiltrating the injured gastric mucosa may induce the emergence of CD44v9 during regeneration of the epithelium. Support or Funding Information This work was supported by NIH 2 R01 DK083402‐06A1 grant, College of Medicine Bridge Funding Program (Zavros), and the University of Cincinnati Graduate School Dean's Fellowship, Albert J. Ryan Fellowship and 2T32GM105526‐04 (Bertaux‐Skeirik).