Humanized mice reveal a macrophage-enriched gene signature defining human lung tissue protection during SARS-CoV-2 infection.

The human immunological mechanisms defining the clinical outcome of SARS-CoV-2 infection remain elusive. This knowledge gap is mostly driven by the lack of appropriate experimental platforms recapitulating human immune responses in a controlled human lung environment. Here, we report a mouse model (i.e., HNFL mice) co-engrafted with human fetal lung xenografts (fLX) and a myeloid-enhanced human immune system to identify cellular and molecular correlates of lung protection during SARS-CoV-2 infection. Unlike mice solely engrafted with human fLX, HNFL mice are protected against infection, severe inflammation, and histopathological phenotypes. Lung tissue protection from infection and severe histopathology associates with macrophage infiltration and differentiation and the upregulation of a macrophage-enriched signature composed of 11 specific genes mainly associated with the type I interferon signaling pathway. Our work highlights the HNFL model as a transformative platform to investigate, in controlled experimental settings, human myeloid immune mechanisms governing lung tissue protection during SARS-CoV-2 infection.

Mast cells regulate CD4+ T-cell differentiation in the absence of antigen presentation.

BACKGROUND: Given their unique capacity for antigen uptake, processing, and presentation, antigen-presenting cells (APCs) are critical for initiating and regulating innate and adaptive immune responses. We have previously shown the role of nicotinamide adenine dinucleotide (NAD+) in T-cell differentiation independently of the cytokine milieu, whereas the precise mechanisms remained unknown. OBJECTIVE: The objective of this study is to further dissect the mechanism of actions of NAD+ and determine the effect of APCs on NAD+-mediated T-cell activation. METHODS: Isolated dendritic cells and bone marrow-derived mast cells (MCs) were used to characterize the mechanisms of action of NAD+ on CD4+ T-cell fate in vitro. Furthermore, NAD+-mediated CD4+ T-cell differentiation was investigated in vivo by using wild-type C57BL/6, MC-/-, MHC class II-/-, Wiskott-Aldrich syndrome protein (WASP)-/-, 5C.C7 recombination-activating gene 2 (Rag2)-/-, and CD11b-DTR transgenic mice. Finally, we tested the physiologic effect of NAD+ on the systemic immune response in the context of Listeria monocytogenes infection. RESULTS: Our in vivo and in vitro findings indicate that after NAD+ administration, MCs exclusively promote CD4+ T-cell differentiation, both in the absence of antigen and independently of major APCs. Moreover, we found that MCs mediated CD4+ T-cell differentiation independently of MHC II and T-cell receptor signaling machinery. More importantly, although treatment with NAD+ resulted in decreased MHC II expression on CD11c+ cells, MC-mediated CD4+ T-cell differentiation rendered mice resistant to administration of lethal doses of L monocytogenes. CONCLUSIONS: Collectively, our study unravels a novel cellular and molecular pathway that regulates innate and adaptive immunity through MCs exclusively and underscores the therapeutic potential of NAD+ in the context of primary immunodeficiencies and antimicrobial resistance.

Epithelialization of mouse ovarian tumor cells originating in the fallopian tube stroma.

Epithelial ovarian carcinoma accounts for 90% of all ovarian cancer and is the most deadly gynecologic malignancy. Recent studies have suggested that fallopian tube fimbriae can be the origin of cells for high-grade serous subtype of epithelial ovarian carcinoma (HGSOC). A mouse HGSOC model with conditional Dicer-Pten double knockout (Dicer-Pten DKO) developed primary tumors, intriguingly, from the fallopian tube stroma. We examined the growth and epithelial phenotypes of the Dicer-Pten DKO mouse tumor cells contributable by each gene knockout. Unlike human ovarian epithelial cancer cells that expressed full-length E-cadherin, the Dicer-Pten DKO stromal tumor cells expressed cleaved E-cadherin fragments and metalloproteinase 2, a mixture of epithelial and mesenchymal markers. Although the Dicer-Pten DKO tumor cells lost the expression of mature microRNAs as expected, they showed high levels of tRNA fragment expression and enhanced AKT activation due to the loss of PTEN function. Introduction of a Dicer1-expressing construct into the DKO mouse tumor cells significantly reduced DNA synthesis and the cell growth rate, with concurrent diminished adhesion and ZO1 epithelial staining. Hence, it is likely that the loss of Dicer promoted mesenchymal-epithelial transition in fallopian tube stromal cells, and in conjunction with Pten loss, further promoted cell proliferation and epithelial-like tumorigenesis.

Elevated CD14 (Cluster of Differentiation 14) and Toll-Like Receptor (TLR) 4 Signaling Deteriorate Periapical Inflammation in TLR2 Deficient Mice.

Apical periodontitis (periapical lesions) is an infection-induced chronic inflammation in the jaw, ultimately resulting in the destruction of apical periodontal tissue. Toll-like receptors (TLRs) are prominent in the initial recognition of pathogens. Our previous study showed that TLR4 signaling is proinflammatory in periapical lesions induced by a polymicrobial endodontic infection. In contrast, the functional role of TLR2 in regulation of periapical tissue destruction is still not fully understood. Using TLR2 deficient (KO), TLR2/TLR4 double deficient (dKO), and wild-type (WT) mice, we demonstrate that TLR2 KO mice are highly responsive to polymicrobial infection-induced periapical lesion caused by over activation of TLR4 signal transduction pathway that resulted in elevation of NF-kB (nuclear factor kappa B) and proinflammatory cytokine production. The altered TLR4 signaling is caused by TLR2 deficiency-dependent elevation of CD14 (cluster of differentiation 14), which is a co-receptor of TLR4. Indeed, neutralization of CD14 strikingly suppresses TLR2 deficiency-dependent inflammation and tissue destruction in vitro and in vivo. Our findings suggest that a network of TLR2, TLR4, and CD14 is a key factor in regulation of polymicrobial dentoalveolar infection and subsequent tissue destruction. Anat Rec, 299:1281-1292, 2016. © 2016 Wiley Periodicals, Inc.

Identification and functional analysis of novel facial patterning genes in the duplicated beak chicken embryo.

Cranial neural crest cells form the majority of the facial skeleton. However exactly when the pattering information and hence jaw identity is established is not clear. We know that premigratory neural crest cells contain a limited amount of information about the lower jaw but the upper jaw and facial midline are specified later by local tissue interactions. The environmental signals leading to frontonasal identity have been explored by our group in the past. Altering the levels of two signaling pathways (Bone Morphogenetic Protein) and retinoic acid (RA) in the chicken embryo creates a duplicated midline on the side of the upper beak complete with egg tooth in place of maxillary derivatives (Lee et al., 2001). Here we analyze the transcriptome 16 h after bead placement in order to identify potential mediators of the identity change in the maxillary prominence. The gene list included RA, BMP and WNT signaling pathway genes as well as transcription factors expressed in craniofacial development. There was also cross talk between Noggin and RA such that Noggin activated the RA pathway. We also observed expression changes in several poorly characterized genes including the upregulation of Peptidase Inhibitor-15 (PI15). We tested the functional effects of PI15 overexpression with a retroviral misexpression strategy. PI15 virus induced a cleft beak analogous to human cleft lip. We next asked whether PI15 effects were mediated by changes in expression of major clefting genes and genes in the retinoid signaling pathway. Expression of TP63, TBX22, BMP4 and FOXE1, all human clefting genes, were upregulated. In addition, ALDH1A2, ALDH1A3 and RA target, RARß were increased while the degradation enzyme CYP26A1 was decreased. Together these changes were consistent with activation of the RA pathway. Furthermore, PI15 retrovirus injected into the face was able to replace RA and synergize with Noggin to induce beak transformations. We conclude that the microarrays have generated a rich dataset containing genes with important roles in facial morphogenesis. Moreover, one of these facial genes, PI15 is a putative clefting gene and is in a positive feedback loop with RA.

NAD+ protects against EAE by regulating CD4+ T-cell differentiation.

CD4(+) T cells are involved in the development of autoimmunity, including multiple sclerosis (MS). Here we show that nicotinamide adenine dinucleotide (NAD(+)) blocks experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, by inducing immune homeostasis through CD4(+)IFNγ(+)IL-10(+) T cells and reverses disease progression by restoring tissue integrity via remyelination and neuroregeneration. We show that NAD(+) regulates CD4(+) T-cell differentiation through tryptophan hydroxylase-1 (Tph1), independently of well-established transcription factors. In the presence of NAD(+), the frequency of T-bet(-/-) CD4(+)IFNγ(+) T cells was twofold higher than wild-type CD4(+) T cells cultured in conventional T helper 1 polarizing conditions. Our findings unravel a new pathway orchestrating CD4(+) T-cell differentiation and demonstrate that NAD(+) may serve as a powerful therapeutic agent for the treatment of autoimmune and other diseases.

MicroRNA-146a and microRNA-155 show tissue-dependent expression in dental pulp, gingival and periodontal ligament fibroblasts in vitro.

MicroRNAs (miRNAs) are small non-coding RNAs showing a tissue-specific expression pattern, and whose function is to suppress protein synthesis. In this study, we hypothesized that expression of miRNAs would differ among fibroblasts from dental pulp (DPF), gingiva (GF) and periodontal ligament (PLF) in vitro. Once established by an explant technique, DPF, GF and PLF were collected for RNA isolation and subjected to a miRNA microarray. Next, cells were stimulated with E. coli lipopolysaccharide (LPS) for 24 h and then collected for RNA isolation. Expression of miR-146a and miR-155 was investigated by qPCR. Microarray screening revealed several miRNAs that showed specifically high expression in at least one of the fibroblast subtypes. These molecules are potentially involved in the regulation of extracellular matrix turnover and production of inflammatory mediators. Microarray analysis showed that both miR-146a and miR-155 were among the miRNAs expressed exclusively in GF. qPCR demonstrated significant upregulation of miR-146a only in GF after LPS stimulation, whereas basal expression of miR-155 was higher in GF than in the other cell subtypes. LPS downregulated the expression of miR-155 only in GF. Our results suggest that the expression and regulation of miR-146a and miR-155 are more pronounced in GF than in DPF and PLF.

Sensory ability in the narwhal tooth organ system.

The erupted tusk of the narwhal exhibits sensory ability. The hypothesized sensory pathway begins with ocean water entering through cementum channels to a network of patent dentinal tubules extending from the dentinocementum junction to the inner pulpal wall. Circumpulpal sensory structures then signal pulpal nerves terminating near the base of the tusk. The maxillary division of the fifth cranial nerve then transmits this sensory information to the brain. This sensory pathway was first described in published results of patent dentinal tubules, and evidence from dissection of tusk nerve connection via the maxillary division of the fifth cranial nerve to the brain. New evidence presented here indicates that the patent dentinal tubules communicate with open channels through a porous cementum from the ocean environment. The ability of pulpal tissue to react to external stimuli is supported by immunohistochemical detection of neuronal markers in the pulp and gene expression of pulpal sensory nerve tissue. Final confirmation of sensory ability is demonstrated by significant changes in heart rate when alternating solutions of high-salt and fresh water are exposed to the external tusk surface. Additional supporting information for function includes new observations of dentinal tubule networks evident in unerupted tusks, female erupted tusks, and vestigial teeth. New findings of sexual foraging divergence documented by stable isotope and fatty acid results add to the discussion of the functional significance of the narwhal tusk. The combined evidence suggests multiple tusk functions may have driven the tooth organ system's evolutionary development and persistence.

Engineering anisotropic biomimetic fibrocartilage microenvironment by bioprinting mesenchymal stem cells in nanoliter gel droplets.

Over the past decade, bioprinting has emerged as a promising patterning strategy to organize cells and extracellular components both in two and three dimensions (2D and 3D) to engineer functional tissue mimicking constructs. So far, tissue printing has neither been used for 3D patterning of mesenchymal stem cells (MSCs) in multiphase growth factor embedded 3D hydrogels nor been investigated phenotypically in terms of simultaneous differentiation into different cell types within the same micropatterned 3D tissue constructs. Accordingly, we demonstrated a biochemical gradient by bioprinting nanoliter droplets encapsulating human MSCs, bone morphogenetic protein 2 (BMP-2), and transforming growth factor ß1 (TGF- ß1), engineering an anisotropic biomimetic fibrocartilage microenvironment. Assessment of the model tissue construct displayed multiphasic anisotropy of the incorporated biochemical factors after patterning. Quantitative real time polymerase chain reaction (qRT-PCR) results suggested genomic expression patterns leading to simultaneous differentiation of MSC populations into osteogenic and chondrogenic phenotype within the multiphasic construct, evidenced by upregulation of osteogenesis and condrogenesis related genes during in vitro culture. Comprehensive phenotypic network and pathway analysis results, which were based on genomic expression data, indicated activation of differentiation related mechanisms, via signaling pathways, including TGF, BMP, and vascular endothelial growth factor.

Current methods for the isolation of extracellular vesicles.

Extracellular vesicles (EVs), including microvesicles and exosomes, are nano- to micron-sized vesicles, which may deliver bioactive cargos that include lipids, growth factors and their receptors, proteases, signaling molecules, as well as mRNA and non-coding RNA, released from the cell of origin, to target cells. EVs are released by all cell types and likely induced by mechanisms involved in oncogenic transformation, environmental stimulation, cellular activation, oxidative stress, or death. Ongoing studies investigate the molecular mechanisms and mediators of EVs-based intercellular communication at physiological and oncogenic conditions with the hope of using this information as a possible source for explaining physiological processes in addition to using them as therapeutic targets and disease biomarkers in a variety of diseases. A major limitation in this evolving discipline is the hardship and the lack of standardization for already challenging techniques to isolate EVs. Technical advances have been accomplished in the field of isolation with improving knowledge and emerging novel technologies, including ultracentrifugation, microfluidics, magnetic beads and filtration-based isolation methods. In this review, we will discuss the latest advances in methods of isolation methods and production of clinical grade EVs as well as their advantages and disadvantages, and the justification for their support and the challenges that they encounter.

Impact of biofluid viscosity on size and sedimentation efficiency of the isolated microvesicles.

Microvesicles are nano-sized lipid vesicles released by all cells in vivo and in vitro. They are released physiologically under normal conditions but their rate of release is higher under pathological conditions such as tumors. Once released they end up in the systemic circulation and have been found and characterized in all biofluids such as plasma, serum, cerebrospinal fluid, breast milk, ascites, and urine. Microvesicles represent the status of the donor cell they are released from and they are currently under intense investigation as a potential source for disease biomarkers. Currently, the "gold standard" for isolating microvesicles is ultracentrifugation, although alternative techniques such as affinity purification have been explored. Viscosity is the resistance of a fluid to a deforming force by either shear or tensile stress. The different chemical and molecular compositions of biofluids have an effect on its viscosity and this could affect movements of the particles inside the fluid. In this manuscript we addressed the issue of whether viscosity has an effect on sedimentation efficiency of microvesicles using ultracentrifugation. We used different biofluids and spiked them with polystyrene beads and assessed their recovery using the Nanoparticle Tracking Analysis. We demonstrate that MVs recovery inversely correlates with viscosity and as a result, sample dilutions should be considered prior to ultracentrifugation when processing any biofluids.

A primer on the current state of microarray technologies.

DNA microarray technology has been used for genome-wide gene expression studies that incorporate molecular genetics and computer science analyses on massive levels. The availability of microarrays permit the simultaneous analysis of tens of thousands of genes for the purposes of gene discovery, disease diagnosis, improved drug development, and therapeutics tailored to specific disease processes. In this chapter, we provide an overview on the current state of common microarray technologies and platforms. Since many genes contribute to normal functioning, research efforts are moving from the search for a disease-specific gene to the understanding of the biochemical and molecular functioning of a variety of genes whose disrupted interaction in complicated networks can lead to a disease state. The field of microarrays has evolved over the past decade and is now standardized with a high level of quality control, while providing a relatively inexpensive and reliable alternative to studying various aspects of gene expression.

Thermal stabilization of tissues and the preservation of protein phosphorylation states for two-dimensional gel electrophoresis.

2-DE is typically capable of discriminating proteins differing by a single phosphorylation or dephosphorylation event. However, a reliable representation of protein phosphorylation states as they occur in vivo requires that both phosphatases and kinases are rapidly and completely inactivated. Thermal stabilization of mouse cerebral cortex homogenates effectively inactivated these enzymes, as evidenced by comparison with unstabilized tissues where abscissal pI shifts were a common feature in 2-D gels. Of the 588 matched proteins separated on 2-D gels comparing stabilized and unstabilized tissues, 53 proteins exhibited greater than twofold differences in spot volume (ANOVA, p<0.05). Phosphoprotein-specific staining was corroborated by the identification of 16 phosphoproteins by nano-LC MS/MS and phosphotyrosine kinase activity assay.

Long-term changes of serum chemokine levels in vaccinated military personnel.

BACKGROUND: Members of the United States Armed Forces receive a series of vaccinations during their course of service. To investigate the influence of multiple vaccinations on innate immunity, we measured concentrations of a panel of immunomodulatory and pro-inflammatory cytokines in serum samples from a group of such individuals. RESULTS: Significantly increased levels of macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta and interleukin 8 (IL-8) were detected. Since these cytokines are known to have anti-human immunodeficiency virus (HIV) activity, we tested the effect of serum from these individuals on HIV-1 infectivity and susceptibility of their peripheral blood mononuclear cells (PBMCs) to HIV-1 infection in vitro. Sera from vaccinated military personnel inhibited, and their PBMCs were partially resistant to, infection by HIV-1 strains tropic to CCR5 (R5), but not to CXCR4 (X4), chemokine receptor. CONCLUSION: These findings demonstrate that increased anti-HIV chemokines can be detected in vaccine recipients up to 68 weeks following immunization.