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NK cells are key innate immune cells that play critical roles in host defense. Although NK cells have been shown to regulate immunity to some infectious diseases,their role in immunity to Trypanosoma congolense has not been investigated. NK cells are vital sources of IFN-gamma and TNF-alpha; two key cytokines that are known to play important roles in resistance to African trypanosomes. In this article,we show that infection with T. congolense leads to increased levels of activated and functional NK cells in multiple tissue compartments. Systemic depletion of NK cells with anti-NK1.1 mAb led to increased parasitemia,which was accompanied by significant reduction in IFN-gamma production by immune cells in the spleens and liver of infected mice. Strikingly,infected NFIL3-/- mice (which genetically lack NK cell development and function) on the normally resistant background were highly susceptible to T. congolense infection. These mice developed fulminating and uncontrolled parasitemia and died significantly earlier (13 ± 1 d) than their wild-type control mice (106 ± 26 d). The enhanced susceptibility of NFIL3-/- mice to infection was accompanied by significantly impaired cytokine (IFN-gamma and TNF-alpha) response by CD3+ T cells in the spleens and liver. Adoptive transfer of NK cells into NFIL3-/- mice before infection rescued them from acute death in a perforin-dependent manner. Collectively,these studies show that NK cells are critical for optimal resistance to T. congolense,and its deficiency leads to enhanced susceptibility in infected mice. View Publication

Tissue inhibitor matrix metalloproteinase-1 (TIMP1) is one of four identified members of the TIMP family. We evaluated the role of TIMP1 in gastric cancer using human and mouse tissues along with gastric organoids and in vitro cell models. Using quantitative real-time RT-PCR,we detected significant overexpression of TIMP1 in the human gastric cancer samples,as compared to normal stomach samples (P {\textless} 0.01). We also detected overexpression of Timp1 in neoplastic gastric lesions of the Tff1-knockout (KO) mice,as compared to normal stomach tissues. Reconstitution of TFF1 in human gastric cancer cell lines led to a significant decrease in the mRNA expression level of TIMP1 (P {\textless} 0.05). In vitro analysis demonstrated that TIMP1 mRNA expression is induced by TNF-alpha and activation of NF-kappaB whereas inhibition of NF-kappaB using BAY11-7082 led to inhibition of NF-kappaB and downregulation of TIMP1. Western blot analysis confirmed the decrease in TIMP1 protein level following reconstitution of TFF1. By using immunofluorescence,we showed nuclear localization of NF-kappaB and expression of TIMP1 in gastric organoids established from the Tff1-KO stomach where reconstitution of Tff1 using recombinant protein led to a notable reduction in the expression of both NF-kappaB and TIMP1. Using EDU assay,as a measure of proliferating cells,we found that TIMP1 promotes cellular proliferation whereas TFF1 reconstitution leads to a significant decrease in cellular proliferation (P {\textless} 0.05). In summary,our findings demonstrate overexpression of TIMP1 in mouse and human gastric cancers through NF-kB-dependent mechanism. We also show that TFF1 suppresses NF-kappaB and inhibits TIMP1-mediated proliferative potential in gastric cancer. View Publication

Antibodies secreted into mucosal barriers serve to protect the host from a variety of pathogens,and are the basis for successful vaccines1. In type I mucosa (such as the intestinal tract),dimeric IgA secreted by local plasma cells is transported through polymeric immunoglobulin receptors2 and mediates robust protection against viruses3,4. However,owing to the paucity of polymeric immunoglobulin receptors and plasma cells,how and whether antibodies are delivered to the type II mucosa represented by the lumen of the lower female reproductive tract remains unclear. Here,using genital herpes infection in mice,we show that primary infection does not establish plasma cells in the lamina propria of the female reproductive tract. Instead,upon secondary challenge with herpes simplex virus 2,circulating memory B cells that enter the female reproductive tract serve as the source of rapid and robust antibody secretion into the lumen of this tract. CD4 tissue-resident memory T cells secrete interferon-gamma,which induces expression of chemokines,including CXCL9 and CXCL10. Circulating memory B cells are recruited to the vaginal mucosa in a CXCR3-dependent manner,and secrete virus-specific IgG2b,IgG2c and IgA into the lumen. These results reveal that circulating memory B cells act as a rapidly inducible source of mucosal antibodies in the female reproductive tract. View Publication

CD4 T cells from HIV-1 infected patients die at excessive rates compared to those from uninfected patients,causing immunodeficiency. We previously identified a dominant negative ligand that antagonizes the TRAIL-dependent pathway of cell death,which we called TRAILshort. Because the TRAIL pathway has been implicated in CD4 T cell death occurring during HIV-1 infection,we used short hairpin RNA knockdown,CRISPR deletion,or Abs specific for TRAILshort to determine the effect of inhibiting TRAILshort on the outcome of experimental acute HIV infection in vitro. Strikingly,all three approaches to TRAILshort deletion/inhibition enhanced HIV-induced death of both infected and uninfected human CD4 T cells. Thus,TRAILshort impacts T cell dynamics during HIV infection,and inhibiting TRAILshort causes more HIV-infected and uninfected bystander cells to die. TRAILshort is,therefore,a host-derived,host-adaptive mechanism to limit the effects of TRAIL-induced cell death. Further studies on the effects of TRAILshort in other disease states are warranted. View Publication

Immune activation may underlie the pathogenesis of irritable bowel syndrome (IBS),but the evidence is conflicting. We examined whether peripheral CD4+ T-cells from IBS patients demonstrated immune activation and changes in cytokine production. To gain mechanistic insight,we examined whether immune activation correlated with psychological stress and changing symptoms over time. IBS patients (n = 29) and healthy volunteers (HV; n = 29) completed symptom and psychological questionnaires. IBS patients had a significant increase in CD4+ T-cells expressing the gut homing marker integrin beta7 (p = 0.023) and lymphoid marker CD62L (p = 0.026) compared to HV. Furthermore,phytohaemagglutinin stimulated CD4+ T-cells from IBS-D patients demonstrated increased TNFalpha secretion when compared to HV (p = 0.044). Increased psychological scores in IBS did not correlate with TNFalpha production,while stress hormones inhibited cytokine secretion from CD4+ T-cells of HV in vitro. IBS symptoms,but not markers of immune activation,decreased over time. CD4+ T-cells from IBS-D patients exhibit immune activation,but this did not appear to correlate with psychological stress measurements or changing symptoms over time. This could suggest that immune activation is a surrogate of an initial trigger and/or ongoing parallel peripheral mechanisms. View Publication

Various preclinical models have been developed to clarify the pathophysiology of prostate cancer (PCa). Traditional PCa cell lines from clinical metastatic lesions,as exemplified by DU-145,PC-3,and LNCaP cells,are useful tools to define mechanisms underlying tumorigenesis and drug resistance. Cell line-based experiments,however,have limitations for preclinical studies because those cells are basically adapted to 2-dimensional monolayer culture conditions,in which the majority of primary PCa cells cannot survive. Recent tissue engineering enables generation of PCa patient-derived xenografts (PDXs) from both primary and metastatic lesions. Compared with fresh PCa tissue transplantation in athymic mice,co-injection of PCa tissues with extracellular matrix in highly immunodeficient mice has remarkably improved the success rate of PDX generation. PDX models have advantages to appropriately recapitulate the molecular diversity,cellular heterogeneity,and histology of original patient tumors. In contrast to PDX models,patient-derived organoid and spheroid PCa models in 3-dimensional culture are more feasible tools for in vitro studies for retaining the characteristics of patient tumors. In this article,we review PCa preclinical model cell lines and their sublines,PDXs,and patient-derived organoid and spheroid models. These PCa models will be applied to the development of new strategies for cancer precision medicine. View Publication

A large body of evidence suggests that B-cell lymphomas with enhanced Myc expression are associated with an aggressive phenotype and poor prognosis,which makes Myc a compelling therapeutic target. Phosphodiesterase 4B (PDE4B),a main hydrolyzer of cyclic AMP (cAMP) in B cells,was shown to be involved in cell survival and drug resistance in diffuse large B cell lymphomas (DLBCL). However,the interrelationship between Myc and PDE4B remains unclear. Here,we first demonstrate the presence of the Myc-PDE4B feed-forward loop,in which Myc and PDE4B mutually reinforce the expression of each other. Next,the combined targeting of Myc and PDE4 synergistically prevented the proliferation and survival of B lymphoma cells in vitro and in a mouse xenograft model. We finally recapitulated this combinatorial effect in Emu-myc transgenic mice; co-inhibition of Myc and PDE4 suppressed lymphomagenesis and restored B cell development to the wild type level that was associated with marked reduction in Myc levels,unveiling the critical role of the Myc-PDE4B amplification loop in the regulation of Myc expression and the pathogenesis of B cell lymphoma. These findings suggest that the disruption of the Myc-PDE4B circuitry can be exploited in the treatment of B cell malignancies. View Publication

Serrated adenocarcinoma,an alternative pathway for colorectal cancer (CRC) development,accounts for 15{\%}-30{\%} of all CRCs and is aggressive and treatment resistant. We show that the expression of atypical protein kinase C zeta (PKCzeta) and PKClambda/iota was reduced in human serrated tumors. Simultaneous inactivation of the encoding genes in the mouse intestinal epithelium resulted in spontaneous serrated tumorigenesis that progressed to advanced cancer with a strongly reactive and immunosuppressive stroma. Whereas epithelial PKClambda/iota deficiency led to immunogenic cell death and the infiltration of CD8+ T cells,which repressed tumor initiation,PKCzeta loss impaired interferon and CD8+ T cell responses,which resulted in tumorigenesis. Combined treatment with a TGF-beta receptor inhibitor plus anti-PD-L1 checkpoint blockade showed synergistic curative activity. Analysis of human samples supported the relevance of these kinases in the immunosurveillance defects of human serrated CRC. These findings provide insight into avenues for the detection and treatment of this poor-prognosis subtype of CRC. View Publication

This study aimed to determine the association between non-high-fat diet-induced obesity- (non-DIO-) associated gut microbiome dysbiosis with gut abnormalities like cellular turnover of intestinal cells,tight junctions,and mucin formation that can impact gut permeability. We used leptin-deficient (Lepob/ob) mice in comparison to C57BL/6J control mice,which are fed on identical diets,and performed comparative and correlative analyses of gut microbiome composition,gut permeability,intestinal structural changes,tight junction-mucin formation,cellular turnover,and stemness genes. We found that obesity impacted cellular turnover of the intestine with increased cell death and cell survival/proliferation gene expression with enhanced stemness,which are associated with increased intestinal permeability,changes in villi/crypt length,and decreased expression of tight junctions and mucus synthesis genes along with dysbiotic gut microbiome signature. Obesity-induced gut microbiome dysbiosis is also associated with abnormal intestinal organoid formation characterized with decreased budding and higher stemness. Results suggest that non-DIO-associated gut microbiome dysbiosis is associated with changes in the intestinal cell death versus cell proliferation homeostasis and functions to control tight junctions and mucous synthesis-regulating gut permeability. View Publication

BACKGROUND Human microRNAs (miRNAs) have diverse functions in biology,and play a role in nearly every biological process. Here we report that miR-520d-5p (520d-5p) causes undifferentiated cancer cells to adopt benign or normal status in vivo in immunodeficient mice via demethylation and P53 upregulation. Further we found that 520-5p causes normal cells to elongate cellular lifetime and mesenchymal stem cell-like status with CD105 positivity. We hypothesized that ectopic 520d-5p expression reduced mutations in undifferentiated type of hepatoma (HLF) cells through synergistic modulation of methylation-related enzymatic expression. METHODS To examine whether there were any changes in mutation status in cells treated with 520d-5p,we performed next generation sequencing (NGS) in HLF cells and human iPSC-derivative cells in pre-mesenchymal stem cell status. We analyzed the data using both genome-wide and individual gene function approaches. RESULTS 520d-5p induced a shift towards a wild type or non-malignant phenotype,which was regulated by nucleotide mutations in both HLF cells and iPSCs. Further,520d-5p reduced mutation levels in both the whole genome and genomic fragment assemblies. CONCLUSIONS Cancer cell genomic mutations cannot be repaired in most contexts. However,these findings suggest that applied development of 520d-5p would allow new approaches to cancer research and improve the quality of iPSCs used in regenerative medicine. View Publication

Mesenchymal stromal cells (MSCs) are multipotent stem cells that participate in tissue repair and posses considerable immunomodulatory potential. MSCs have been shown to promote allograft survival,yet the mechanisms behind this phenomenon have not been fully defined. Here,we investigate the capacity of MSCs to suppress the allogeneic immune response by secreting the pleiotropic molecule hepatocyte growth factor (HGF). Using an in vivo mouse model of corneal transplantation,we report that MSCs promote graft survival in an HGF-dependent manner. Moreover,our data indicate that topically administered recombinant HGF (1) suppresses antigen-presenting cell maturation in draining lymphoid tissue,(2) limits T-helper type-1 cell generation,(3) decreases inflammatory cell infiltration into grafted tissue,and (4) is itself sufficient to promote transplant survival. These findings have potential translational implications for the development of HGF-based therapeutics. Stem Cells Translational Medicine 2019. View Publication

Antagonists of the 5-hydroxytryptamine (serotonin) 3 receptor (5-HT3R) have anti-inflammatory and anti-apoptotic activities,but the detailed,underlying mechanisms are not well understood. We focused on anti-apoptotic activities via 5-HT3R signaling to clarify the underlying mechanisms. Mice were administered 5-fluorouracil (5-FU),which induced apoptosis in intestinal epithelial cells. Coadministration with 5-HT3R antagonists or agonists tended to decrease or increase the number of apoptotic cells,respectively. In serotonin 3A receptor (5-HT3AR) null (HTR3A-/-) mice,the number of apoptotic cells induced by 5-FU was decreased compared with that in wild-type (WT) mice. Bone marrow (BM) transplantation was performed to determine if BM-derived immune cells regulated 5-FU-induced apoptosis,but they were found to be unrelated to this process. Data from 5-HT3AR/enhanced green fluorescent protein reporter mice revealed that 50{\%} of enterochromaffin (EC) cells expressed 5-HT3AR,but the number of apoptotic cells induced by 5-FU in the intestinal crypt organoids of HTR3A-/- mice was not altered compared with WT mice. In contrast,plasma 5-HT concentrations in WT mice but not in HTR3A-/- mice administered 5-FU were increased significantly. In conclusion,5-HT3R signaling may enhance 5-HT release,possibly from EC cells intravascularly,or paracrine,resulting in increases in plasma 5-HT concentration,which in turn,enhances apoptotic activities induced by 5-FU.-Mikawa,S.,Kondo,M.,Kaji,N.,Mihara,T.,Yoshitake,R.,Nakagawa,T.,Takamoto,M.,Nishimura,R.,Shimada,S.,Ozaki,H.,Hori,M. Serotonin 3 receptor signaling regulates 5-fluorouracil-mediated apoptosis indirectly via TNF-alpha production by enhancing serotonin release from enterochromaffin cells. View Publication