Type II taste cells participate in mucosal immune surveillance.

The oral microbiome is second only to its intestinal counterpart in diversity and abundance, but its effects on taste cells remains largely unexplored. Using single-cell RNASeq, we found that mouse taste cells, in particular, sweet and umami receptor cells that express taste 1 receptor member 3 (Tas1r3), have a gene expression signature reminiscent of Microfold (M) cells, a central player in immune surveillance in the mucosa-associated lymphoid tissue (MALT) such as those in the Peyer's patch and tonsils. Administration of tumor necrosis factor ligand superfamily member 11 (TNFSF11; also known as RANKL), a growth factor required for differentiation of M cells, dramatically increased M cell proliferation and marker gene expression in the taste papillae and in cultured taste organoids from wild-type (WT) mice. Taste papillae and organoids from knockout mice lacking Spib (SpibKO), a RANKL-regulated transcription factor required for M cell development and regeneration on the other hand, failed to respond to RANKL. Taste papillae from SpibKO mice also showed reduced expression of NF-κB signaling pathway components and proinflammatory cytokines and attracted fewer immune cells. However, lipopolysaccharide-induced expression of cytokines was strongly up-regulated in SpibKO mice compared to their WT counterparts. Like M cells, taste cells from WT but not SpibKO mice readily took up fluorescently labeled microbeads, a proxy for microbial transcytosis. The proportion of taste cell subtypes are unaltered in SpibKO mice; however, they displayed increased attraction to sweet and umami taste stimuli. We propose that taste cells are involved in immune surveillance and may tune their taste responses to microbial signaling and infection.

Nkx2-2 expressing taste cells in endoderm-derived taste papillae are committed to the type III lineage.

In mammals, multiple cell-signaling pathways and transcription factors regulate development of the embryonic taste system and turnover of taste cells in the adult stage. Using single-cell RNA-Seq of mouse taste cells, we found that the homeobox-containing transcription factor Nkx2-2, a target of the Sonic Hedgehog pathway and a key regulator of the development and regeneration of multiple cell types in the body, is highly expressed in type III taste cells but not in type II or taste stem cells. Using in situ hybridization and immunostaining, we confirmed that Nkx2-2 is expressed specifically in type III taste cells in the endoderm-derived circumvallate and foliate taste papillae but not in the ectoderm-derived fungiform papillae. Lineage tracing revealed that Nkx2-2-expressing cells differentiate into type III, but not type II or type I cells in circumvallate and foliate papillae. Neonatal Nkx2-2-knockout mice did not express key type III taste cell marker genes, while the expression of type II and type I taste cell marker genes were unaffected in these mice. Our findings indicate that Nkx2-2-expressing cells are committed to the type III lineage and that Nkx2-2 may be critical for the development of type III taste cells in the posterior tongue, thus illustrating a key difference in the mechanism of type III cell lineage specification between ectoderm- and endoderm-derived taste fields.

Gli3 is a negative regulator of Tas1r3-expressing taste cells.

Mouse taste receptor cells survive from 3-24 days, necessitating their regeneration throughout adulthood. In anterior tongue, sonic hedgehog (SHH), released by a subpopulation of basal taste cells, regulates transcription factors Gli2 and Gli3 in stem cells to control taste cell regeneration. Using single-cell RNA-Seq we found that Gli3 is highly expressed in Tas1r3-expressing taste receptor cells and Lgr5+ taste stem cells in posterior tongue. By PCR and immunohistochemistry we found that Gli3 was expressed in taste buds in all taste fields. Conditional knockout mice lacking Gli3 in the posterior tongue (Gli3CKO) had larger taste buds containing more taste cells than did control wild-type (Gli3WT) mice. In comparison to wild-type mice, Gli3CKO mice had more Lgr5+ and Tas1r3+ cells, but fewer type III cells. Similar changes were observed ex vivo in Gli3CKO taste organoids cultured from Lgr5+ taste stem cells. Further, the expression of several taste marker and Gli3 target genes was altered in Gli3CKO mice and/or organoids. Mirroring these changes, Gli3CKO mice had increased lick responses to sweet and umami stimuli, decreased lick responses to bitter and sour taste stimuli, and increased glossopharyngeal taste nerve responses to sweet and bitter compounds. Our results indicate that Gli3 is a suppressor of stem cell proliferation that affects the number and function of mature taste cells, especially Tas1r3+ cells, in adult posterior tongue. Our findings shed light on the role of the Shh pathway in adult taste cell regeneration and may help devise strategies for treating taste distortions from chemotherapy and aging.

Whole transcriptome profiling of taste bud cells.

Analysis of single-cell RNA-Seq data can provide insights into the specific functions of individual cell types that compose complex tissues. Here, we examined gene expression in two distinct subpopulations of mouse taste cells: Tas1r3-expressing type II cells and physiologically identified type III cells. Our RNA-Seq libraries met high quality control standards and accurately captured differential expression of marker genes for type II (e.g. the Tas1r genes, Plcb2, Trpm5) and type III (e.g. Pkd2l1, Ncam, Snap25) taste cells. Bioinformatics analysis showed that genes regulating responses to stimuli were up-regulated in type II cells, while pathways related to neuronal function were up-regulated in type III cells. We also identified highly expressed genes and pathways associated with chemotaxis and axon guidance, providing new insights into the mechanisms underlying integration of new taste cells into the taste bud. We validated our results by immunohistochemically confirming expression of selected genes encoding synaptic (Cplx2 and Pclo) and semaphorin signalling pathway (Crmp2, PlexinB1, Fes and Sema4a) components. The approach described here could provide a comprehensive map of gene expression for all taste cell subpopulations and will be particularly relevant for cell types in taste buds and other tissues that can be identified only by physiological methods.

MOAP-1 Mediates Fas-Induced Apoptosis in Liver by Facilitating tBid Recruitment to Mitochondria.

Fas apoptotic signaling regulates diverse physiological processes. Acute activation of Fas signaling triggers massive apoptosis in liver. Upon Fas receptor stimulation, the BH3-only protein Bid is cleaved into the active form, tBid. Subsequent tBid recruitment to mitochondria, which is facilitated by its receptor MTCH2 at the outer mitochondrial membrane (OMM), is a critical step for commitment to apoptosis via the effector proteins Bax or Bak. MOAP-1 is a Bax-binding protein enriched at the OMM. Here, we show that MOAP-1-deficient mice are resistant to Fas-induced hepatocellular apoptosis and lethality. In the absence of MOAP-1, mitochondrial accumulation of tBid is markedly impaired. MOAP-1 binds to MTCH2, and this interaction appears necessary for MTCH2 to engage tBid. These findings reveal a role for MOAP-1 in Fas signaling in the liver by promoting MTCH2-mediated tBid recruitment to mitochondria.

Discovery of PAT-1102, a novel, potent and orally active histone deacetylase inhibitor with antitumor activity in cancer mouse models.

AIM: Histone deacetylase (HDAC) inhibitors are a class of drugs that modulate transcriptional activity in cells and are known to induce cell-cycle arrest and angiogenesis, the major components of tumor cell proliferation. The aim of the present study was to characterize a novel hydroxamic acid-based HDAC inhibitor, PAT-1102, and determine its efficacy and tolerability in pre-clinical models. MATERIALS AND METHODS: HDAC enzyme inhibition was measured using HeLa cell nuclear extracts, and recombinant HDAC enzymes. Antiproliferative activity was assessed in a panel of cancer cell lines. Histone hyper-acetylation status and p21 induction were assessed in HeLa cells by immunoblotting. The effect on apoptosis was tested by caspase-3 activation and detection of cleaved poly-ADP ribose polymerase (PARP). Single-dose pharmacokinetics of the compound were assessed in BALB/c mice following oral and intravenous administration. Antitumor efficacy was evaluated in tumor-bearing mice established from lung and colorectal cancer cells (A549 and HCT116, respectively). RESULTS: PAT-1102 demonstrated potent HDAC-inhibitory activity and growth-inhibitory properties against a panel of cancer cell lines. The optimized compound PAT-1102 exhibits good aqueous solubility, metabolic stability and a favorable pharmacokinetic profile. Once-daily oral administration of PAT-1102 resulted in significant antitumor activity and was well-tolerated in mice. CONCLUSION: Our results indicate that PAT-1102 is a novel, potent, orally available HDAC inhibitor with antiproliferative activity against several human cancer cell lines and antitumor activity in mouse xenograft models. Based on the pre-clinical efficacy and safety profile of PAT-1102, the compound demonstrates significant potential for evaluation as a novel drug candidate for cancer therapy.

Fcγ receptor I alpha chain (CD64) expression in macrophages is critical for the onset of meningitis by Escherichia coli K1.

Neonatal meningitis due to Escherichia coli K1 is a serious illness with unchanged morbidity and mortality rates for the last few decades. The lack of a comprehensive understanding of the mechanisms involved in the development of meningitis contributes to this poor outcome. Here, we demonstrate that depletion of macrophages in newborn mice renders the animals resistant to E. coli K1 induced meningitis. The entry of E. coli K1 into macrophages requires the interaction of outer membrane protein A (OmpA) of E. coli K1 with the alpha chain of Fcγ receptor I (FcγRIa, CD64) for which IgG opsonization is not necessary. Overexpression of full-length but not C-terminal truncated FcγRIa in COS-1 cells permits E. coli K1 to enter the cells. Moreover, OmpA binding to FcγRIa prevents the recruitment of the γ-chain and induces a different pattern of tyrosine phosphorylation of macrophage proteins compared to IgG2a induced phosphorylation. Of note, FcγRIa(-/-) mice are resistant to E. coli infection due to accelerated clearance of bacteria from circulation, which in turn was the result of increased expression of CR3 on macrophages. Reintroduction of human FcγRIa in mouse FcγRIa(-/-) macrophages in vitro increased bacterial survival by suppressing the expression of CR3. Adoptive transfer of wild type macrophages into FcγRIa(-/-) mice restored susceptibility to E. coli infection. Together, these results show that the interaction of FcγRI alpha chain with OmpA plays a key role in the development of neonatal meningitis by E. coli K1.

Inhibition of ubiquitin-mediated degradation of MOAP-1 by apoptotic stimuli promotes Bax function in mitochondria.

The multidomain proapoptotic protein Bax of the Bcl-2 family is a central regulator for controlling the release of apoptogenic factors from mitochondria. Recent evidence suggests that the Bax-associating protein MOAP-1 may act as an effector for promoting Bax function in mitochondria. Here, we report that MOAP-1 protein is rapidly up-regulated by multiple apoptotic stimuli in mammalian cells. MOAP-1 is a short-lived protein (t(1/2) approximately 25 min) that is constitutively degraded by the ubiquitin-proteasome system. Induction of MOAP-1 by apoptotic stimuli ensues through inhibition of its polyubiquitination process. Elevation of MOAP-1 levels sensitizes cells to apoptotic stimuli and promotes recombinant Bax-mediated cytochrome c release from isolated mitochondria. Mitochondria depleted of short-lived proteins by cycloheximide (CHX) become resistant to Bax-mediated cytochrome c release. Remarkably, incubation of these mitochondria with in vitro-translated MOAP-1 effectively restores the cytochrome c releasing effect of recombinant Bax. We propose that apoptotic stimuli can facilitate the proapoptotic function of Bax in mitochondria through stabilization of MOAP-1.

Inhibition of apoptosis by Escherichia coli K1 is accompanied by increased expression of BclXL and blockade of mitochondrial cytochrome c release in macrophages.

Escherichia coli K1 survival in the blood is a critical step for the onset of meningitis in neonates. Therefore, the circulating bacteria are impelled to avoid host defense mechanisms by finding a niche to survive and multiply. Our recent studies have shown that E. coli K1 enters and survives in both monocytes and macrophages in the newborn rat model of meningitis as well as in macrophage cell lines. Here we demonstrate that E. coli K1 not only extends the survival of human and murine infected macrophage cell lines but also renders them resistant to apoptosis induced by staurosporine. Macrophages infected with wild-type E. coli expressing outer membrane protein A (OmpA), but not with OmpA- E. coli, are resistant to DNA fragmentation and phosphatidylserine exposure induced by staurosporine. Infection with OmpA+ E. coli induces the expression of Bcl(XL), an antiapoptotic protein, both at the mRNA level as assessed by gene array analysis and at the protein level as evaluated by immunoblotting. OmpA- E. coli infection of macrophages induced the release of cytochrome c from mitochondria into the cytosol and the activation of caspases 3, 6, and 9, events that were significantly blocked in OmpA+ E. coli-infected macrophages. In addition, OmpA+ E. coli-infected cells were resistant to a decrease in the transmembrane potential of mitochondria induced by staurosporine as measured by the MitoCapture fluorescence technique. Complementation of OmpA- E. coli with a plasmid containing the ompA gene restored the ability of OmpA- E. coli to inhibit the apoptosis of infected macrophages, further demonstrating that E. coli OmpA expression is critical for inducing macrophage survival and thereby finding a safe haven for its growth.

Entry and intracellular replication of Escherichia coli K1 in macrophages require expression of outer membrane protein A.

Interactions between Escherichia coli K1, which causes meningitis in neonates, and macrophages have not been explored well. In this study we found that E. coli K1 was able to enter, survive, and replicate intracellularly in both murine and human macrophage cell lines, as well as in monocytes and macrophages of newborn rats. In addition, we demonstrated that OmpA (+) E. coli also enters and replicates in human peripheral blood monocytes in vitro. Outer membrane protein A (OmpA) expression on E. coli contributes to binding to macrophages, phagocytosis, and survival within macrophages. Opsonization with either complement proteins or antibody is not required for uptake and survival of the bacteria within the macrophages. Transmission electron microscopy and immunocytochemistry studies with the infected macrophages indicated that OmpA(+) E. coli multiplies enormously in a single phagosome and bursts the cell. Internalization of OmpA(+) E. coli by RAW 264.7 cells occurred by both actin- and microtubule-dependent processes, which are independent of RGD-mediated integrin receptors. Internalization and intracellular survival within phagocytic cells thus may play an important role in the development of bacteremia, which is crucial for E. coli crossing of the blood-brain barrier.

Cloning and expression of the Escherichia coli K1 outer membrane protein A receptor, a gp96 homologue.

Escherichia coli is one of the most common gram-negative bacteria that cause meningitis in neonates. Our previous studies have shown that outer membrane protein A (OmpA) of E. coli interacts with a 95-kDa human brain microvascular endothelial cell (HBMEC) glycoprotein, Ecgp, for invasion. Here, we report the identification of a gene that encodes Ecgp by screening of an HBMEC cDNA expression library as well as by 5' rapid amplification of cDNA ends. The sequence of the Ecgp gene shows that it is highly similar to gp96, a tumor rejection antigen-1, and contains an endoplasmic reticulum retention signal, KDEL. Overexpression of either Ecgp or gp96 in both HBMECs and CHO cells increases E. coli binding and invasion. We further show that Ecgp gene-transfected HBMECs express Ecgp on the cell surface despite the presence of the KDEL motif. Northern blot analysis of total RNA from various eukaryotic cells indicates that Ecgp is significantly expressed in HBMECs. Recombinant His-tagged Ecgp blocked E. coli invasion efficiently by binding directly to the bacteria. These results suggest that OmpA of E. coli K1 interacts with a gp96-like molecule on HBMECs for invasion.