Social behaviors are crucial to all mammals. Although the prelimbic cortex (PL, part of medial prefrontal cortex) has been implicated in social behavior, it is not clear which neurons are relevant or how they contribute. We found that PL contains anatomically and molecularly distinct subpopulations that target three downstream regions that have been implicated in social behavior: the nucleus accumbens (NAc), amygdala, and ventral tegmental area. Activation of NAc-projecting PL neurons (PL-NAc), but not the other subpopulations, decreased the preference for a social target. To determine what information PL-NAc neurons convey, we selectively recorded from them and found that individual neurons were active during social investigation, but only in specific spatial locations. Spatially specific manipulation of these neurons bidirectionally regulated the formation of a social-spatial association. Thus, the unexpected combination of social and spatial information within the PL-NAc may contribute to social behavior by supporting social-spatial learning.

Abstract

BACKGROUND:

Interleukin (IL)-25 is a member of the IL-17 family which can promote and augment T-helper type (Th) 2 responses. The expression of IL-25 and its cognate receptor, IL-25 receptor (IL-25R), is upregulated and correlated with disease activity in Th2-associated diseases.

OBJECTIVE:

To examine the expression and function of IL-25 in cutaneous T-cell lymphoma (CTCL).

METHODS:

Expression and localization of IL-25 in lesional skin was investigated using immunohistochemistry. The effect of various cytokines on IL-25 production from normal human epidermal keratinocytes was assessed by quantitative reverse-transcription real-time polymerase chain reaction. Serum IL-25 levels were measured by enzyme-linked immunosorbent assay. The direct effect of IL-25 on tumor cells was also examined using CTCL cell lines and peripheral blood mononuclear cells in Sézary syndrome patients.

RESULTS:

IL-25 expression was increased in epidermal keratinocytes in lesional skin of CTCL. Th2 cytokines, IL-4 and IL-13, and periostin induced IL-25 expression by normal human epidermal keratinocytes. Serum IL-25 levels were increased in patients with advanced CTCL (stage IIB-IV) and correlated with serum lactate dehydrogenase levels. MyLa cells expressed IL-25R and its expression was augmented by stimulation with IL-25. IL-25 enhanced IL-13 production from MyLa cells via phosphorylation of STAT6. Peripheral blood mononuclear cells from one patient with Sézary syndrome expressed IL-25R and showed increase of IL-13 production by IL-25.

CONCLUSIONS:

Th2 cytokines highly expressed in CTCL lesional skin induce IL-25 production by epidermal keratinocytes, which may in turn lead to formation of Th2-dominant microenvironment through the direct induction of IL-13 by tumor cells. 

Microglia and macrophage cells are the primary producers of cytokines in response to neuroinflammatory processes. But these cytokines are also produced by other glial cells, endothelial cells, and neurons. It is essential to identify the cells that produce these cytokines to target their different levels of activation. We used dual RNAscope^® fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) techniques to visualize the mRNA expression pattern of pro- and anti-inflammatory cytokines in microglia/macrophages cells. Using these methods, we can associate one mRNA to specific cell types when combining with different cellular markers by immunofluorescence. Results from RNAscope^® probes IL-1β, TNFα, TGFβ, IL-10 or Arg1, showed colocalization with antibodies for microglia/macrophage cells. These target probes showed adequate sensitivity and specificity to detect mRNA expression. New FISH detection techniques combined with immunohistochemical techniques will help to jointly determine the protein and mRNA localization, as well as provide reliable quantification of the mRNA expression levels.

The favorable features of high-risk human papillomavirus (HPV) in the head and neck are limited to those harboring transcriptionally-active HPV, which occur predominantly in the oropharynx (OP). Factors rendering the OP susceptible to HPV oncogenesis are largely unexplored. The role of cytokeratin 7 (CK7) in predisposition to HPV and cancer in the cervix has been evaluated. However, its significance in the H&N is unknown. CK7 immunohistochemistry was performed on a tissue microarray cohort of OP and non-oropharyngeal (NOP) squamous cell carcinomas (SCC) with known clinical follow-up and HPV E6/7 mRNA status. Expression was graded based on the distribution (1 ≤ 33%, 2 = 33-66%, 3 ≥ 66%) and intensity (1 = weak, 2 = strong) with combined score of ≥ 2 considered positive. Survival analysis was performed. Seventy-four NOPSCCs and 204 OPSCCs were studied. HPV was positive in 2.7% of NOPSCCs and 70.9% of OPSCCs. CK7 was positive in 23.0% of OPSCCs and 14.8% of NOPSCCs (p = 0.2), and in 24.1% of HPV positive versus 17.2% of negative patients (p = 0.2). There was no correlation with age, race, gender, HPV status, histologic type, tumor subsite, treatment, stage, or co-morbidities, and CK7 expression was not significantly associated with overall or disease specific survival. In our series, CK7 is positive in ~ 25% of H&N SCCs, although usually only focally. While CK7 has been suspected to be overexpressed selectively in HPV-related OPSCCs due to their origination from tonsillar crypt epithelium, we did not find any significant difference by anatomic H&N subsite, nor by HPV status, for its expression and found no association with patient survival.

The histone H3 lysine 27 (H3K27) demethylase Kdm6b (Jmjd3) can promote cellular differentiation, however its physiological functions in neurons remain to be fully determined. We studied the expression and function of Kdm6b in differentiating granule neurons of the developing postnatal mouse cerebellum. At postnatal day 7, Kdm6b is expressed throughout the layers of the developing cerebellar cortex, but its expression is upregulated in newborn cerebellar granule neurons (CGNs). Atoh1-Cre mediated conditional knockout of Kdm6b in CGN precursors either alone or in combination with Kdm6a did not disturb the gross morphological development of the cerebellum. Furthermore, RNAi-mediated knockdown of Kdm6b in cultured CGN precursors did not alter the induced expression of early neuronal marker genes upon cell cycle exit. By contrast, knockdown of Kdm6b significantly impaired the induction of a mature neuronal gene expression program, which includes gene products required for functional synapse maturation. Loss of Kdm6b also impaired the ability of Brain-Derived Neurotrophic Factor (BDNF) to induce expression of Grin2c and Tiam1 in maturing CGNs. Taken together, these data reveal a previously unknown role for Kdm6b in the postmitotic stages of CGN maturation and suggest that Kdm6b may work, at least in part, by a transcriptional mechanism that promotes gene sensitivity to regulation by BDNF.

The current study aimed to understand the developmental mechanisms regulating bud tip progenitor cells in the human fetal lung, which are present during branching morphogenesis, and to use this information to induce a bud tip progenitor-like population from human pluripotent stem cells (hPSCs) in vitro. We identified cues that maintained isolated human fetal lung epithelial bud tip progenitor cells in vitro and induced three-dimensional hPSC-derived organoids with bud tip-like domains. Bud tip-like domains could be isolated, expanded, and maintained as a nearly homogeneous population. Molecular and cellular comparisons revealed that hPSC-derived bud tip-like cells are highly similar to native lung bud tip progenitors. hPSC-derived epithelial bud tip-like structures survived in vitro for over 16 weeks, could be easily frozen and thawed, maintained multilineage potential, and successfully engrafted into the airways of immunocompromised mouse lungs, where they persisted for up to 6 weeks and gave rise to several lung epithelial lineages.

Large-scale transcriptome sequencing efforts have vastly expanded the catalog of long non-coding RNAs (lncRNAs) with varying evolutionary conservation, lineage expression, and cancer specificity. Here, we functionally characterize a novel ultraconserved lncRNA, THOR (ENSG00000226856), which exhibits expression exclusively in testis and a broad range of human cancers. THOR knockdown and overexpression in multiple cell lines and animal models alters cell or tumor growth supporting an oncogenic role. We discovered a conserved interaction of THOR with IGF2BP1 and show that THOR contributes to the mRNA stabilization activities of IGF2BP1. Notably, transgenic THOR knockout produced fertilization defects in zebrafish and also conferred a resistance to melanoma onset. Likewise, ectopic expression of human THOR in zebrafish accelerated the onset of melanoma. THOR represents a novel class of functionally important cancer/testis lncRNAs whose structure and function have undergone positive evolutionary selection.

Maintaining cell fate relies on robust mechanisms that prevent the differentiation of specified cells into other cell types. This is especially critical during embryogenesis, when extensive cell proliferation, patterning, and migration events take place. Here we show that vertebrate primordial germ cells (PGCs) are protected from reprogramming into other cell types by the RNA-binding protein Dead end (Dnd). PGCs knocked down for Dnd lose their characteristic morphology and adopt various somatic cell fates. Concomitantly, they gain a gene expression profile reflecting differentiation into cells of different germ layers, in a process that we could direct by expression of specific cell-fate determinants. Importantly, we visualized these events within live zebrafish embryos, which provide temporal information regarding cell reprogramming. Our results shed light on the mechanisms controlling germ cell fate maintenance and are relevant for the formation of teratoma, a tumor class composed of cells from more than one germ layer.

The attenuation of ancestral pro-regenerative pathways may explain why humans do not efficiently regenerate damaged organs. Vertebrate lineages that exhibit robust regeneration, including the teleost zebrafish, provide insights into the maintenance of adult regenerative capacity. Using established models of spinal cord, heart, and retina regeneration, we discovered that zebrafish Treg-like (zTreg) cells rapidly homed to damaged organs. Conditional ablation of zTreg cells blocked organ regeneration by impairing precursor cell proliferation. In addition to modulating inflammation, infiltrating zTreg cells stimulated regeneration through interleukin-10-independent secretion of organ-specific regenerative factors (Ntf3: spinal cord; Nrg1: heart; Igf1: retina). Recombinant regeneration factors rescued the regeneration defects associated with zTreg cell depletion, whereas Foxp3a-deficient zTreg cells infiltrated damaged organs but failed to express regenerative factors. Our data delineate organ-specific roles for Treg cells in maintaining pro-regenerative capacity that could potentially be harnessed for diverse regenerative therapies.

ABSTRACT

Triple-negative breast cancer (TNBC) represents a more aggressive and difficult subtype of breast cancer where responses to chemotherapy occur, but toxicity is significant and resistance often follows. Immunotherapy has shown promising results in various types of cancer, including breast cancer. Here, we investigated a new combination strategy where histone deacetylase inhibitors (HDACi) are applied with immune checkpoint inhibitors to improve immunotherapy responses in TNBC.

Testing different epigenetic modifiers, we focused on the mechanisms underlying HDACi as priming modulators of immunotherapy. Tumor cells were co-cultured with human peripheral blood mononuclear cells (PBMCs) and flow cytometric immunophenotyping was performed to define the role of epigenetic priming in promoting tumor antigen presentation and immune cell activation. We found that HDACi up-regulate PD-L1 mRNA and protein expression in a time-dependent manner in TNBC cells, but not in hormone responsive cells. Focusing on TNBC, HDACi up-regulated PD-L1 and HLA-DR on tumor cells when co-cultured with PBMCs and down-regulated CD4+ Foxp3+ Treg in vitro. HDACi significantly enhanced the in vivo response to PD-1/CTLA-4 blockade in the triple-negative 4T1 breast cancer mouse model, the only currently available experimental system with functional resemblance to human TNBC. This resulted in a significant decrease in tumor growth and increased survival, associated with increased T cell tumor infiltration and a reduction in CD4+Foxp3+ T cells in the tumor microenvironment. Overall, our results suggest a novel role for HDAC inhibition in combination with immune checkpoint inhibitors and identify a promising therapeutic strategy, supporting its further clinical evaluation for TNBC treatment.

Investigating the expression of RNAs that differ by short or single nucleotide sequences at a single-cell level in tissue has been limited by the sensitivity and specificity of in situ hybridization (ISH) techniques. Detection of short isoform-specific sequences requires RNA isolation for PCR analysis-an approach that loses the regional and cell-type-specific distribution of isoforms. Having the capability to distinguish the differential expression of RNA variants in tissue is critical because alterations in mRNA splicing and editing, as well as coding single nucleotide polymorphisms, have been associated with numerous cancers, neurological and psychiatric disorders. Here we introduce a novel highly sensitive single-probe colorimetric/fluorescent ISH approach that targets short exon/exon RNA splice junctions using single-pair oligonucleotide probes (~ 50 bp). We use this approach to investigate, with single-cell resolution, the expression of four transcripts encoding the neuregulin (NRG) receptor ErbB4 that differ by alternative splicing of exons encoding two juxtamembrane (JMa/JMb) and two cytoplasmic (CYT-1/CYT-2) domains that alter receptor stability and signaling modes, respectively. By comparing ErbB4 hybridization on sections from wild-type and ErbB4 knockout mice (missing exon 2), we initially demonstrate that single-pair probes provide the sensitivity and specificity to visualize and quantify the differential expression of ErbB4 isoforms. Using cell-type-specific GFP reporter mice, we go on to demonstrate that expression of ErbB4 isoforms differs between neurons and oligodendrocytes, and that this differential expression of ErbB4 isoforms is evolutionarily conserved to humans. This single-pair probe ISH approach, known as BaseScope, could serve as an invaluable diagnostic tool to detect alternative spliced isoforms, and potentially single base polymorphisms, associated with disease.

Abstract

BACKGROUND:

DUOX2 and DUOXA2 form the predominant H2O2-producing system in human colorectal mucosa. Inflammation, hypoxia, and 5-aminosalicylic acid increase H2O2 production, supporting innate defense and mucosal healing. Thiocyanate reacts with H2O2 in the presence of lactoperoxidase (LPO) to form hypothiocyanate (OSCN-), which acts as a biocide and H2O2 scavenging system to reduce damage during inflammation. We aimed to discover the organization of Duox2, Duoxa2, and Lpo expression in colonic crypts of Lieberkühn (intestinal glands) of mice and how distributions respond to dextran sodium sulfate (DSS)-induced colitis and subsequent mucosal regeneration.

METHODS:

We studied tissue from DSS-exposed mice and human biopsies using in situ hybridization, reverse transcription quantitative polymerase chain reaction, and cDNA microarray analysis.

RESULTS:

Duox2 mRNA expression was mostly in the upper crypt quintile while Duoxa2 was more apically focused. Most Lpo mRNA was in the basal quintile, where stem cells reside. Duox2 and Duoxa2 mRNA were increased during the induction and resolution of DSS colitis, while Lpo expression did not increase during the acute phase. Patterns of Lpo expression differed from Duox2 in normal, inflamed, and regenerative mouse crypts (P < 0.001). We found no evidence of LPO expression in the human gut.

CONCLUSIONS:

The spatial and temporal separation of H2O2-consuming and -producing enzymes enables a thiocyanate- H2O2 "scavenging" system in murine intestinal crypts to protect the stem/proliferative zones from DNA damage, while still supporting higher H2O2 concentrations apically to aid mucosal healing. The absence of LPO expression in the human gut suggests an alternative mechanism or less protection from DNA damage during H2O2-driven mucosal healing.