The COVID-19 pandemic continues to affect the world in 2025. The rapid mutation of SARS-CoV-2 results in breakthrough infections and diminishes the efficacy of vaccines and anti-viral drugs. The severity of the disease varies across different variants, and the underlying mechanisms driving these differences remain unclear. This study explores the relationship between different Spike variants and cytotoxicity, aiming to determine whether the humanized decoy receptor ACE2-Fc can neutralize spikes from diverse variants, offering a solution to overcome rapid mutating SARS-CoV-2 induced immune escape. We co-cultured 293 T-ACE2 cells with 293 T cells transfected with various Spike protein variants or used H1650-ACE2 cells transfected with these Spike variants. This allowed us to observe the effects of different Spike mutations, specifically focusing on cell fusion, cytotoxicity, and cytokine release from human peripheral blood mononuclear cells. Flow cytometry is employed to determine if ACE2-Fc can recognize different Spike variants. We also assess the ability of ACE2-Fc to inhibit infection, cell fusion, cytotoxicity, and cytokine release through pseudovirus infections or Spike protein transfections. Additionally, we use actual viruses from SARS-CoV-2 patients to validate the impacts of Spike mutations and the effectiveness of ACE2-Fc. Furthermore, human plasma is utilized to evaluate ACE2-Fc’s capability to inhibit Spike-induced clot formation. We found that different Spike variants, particularly those with enhancements at the S2' site, increased cell–cell fusion capability, which correlated positively with cytotoxicity and cytokine IL-6 and TNF-α released from PBMCs. ACE2-Fc recognized spikes from wide-range of variants, including wild type, Alpha, Delta, Delta plus, Lambda, BA.2, BA.2.75, BA.5, BF.7, BQ.1, XBB.1, JN.1, KP.2, and KP.3, and effectively prevented these spike-expressing pseudo-viruses from entering host cells. Crucially, ACE2-Fc can prevent spike-induced cell fusion, thereby reducing subsequent cytotoxicity and the release of IL-6 and TNF-α from PBMCs. ACE2-Fc also effectively reduces plasma clot formation induced by trimeric spike proteins. These findings demonstrated that ACE2-Fc could effectively combat the infection of rapidly mutating SARS-CoV-2, providing a potential solution to overcome immune evasion.

Introduction:Idiopathic pulmonary fibrosis is a progressive lung disease with a poor prognosis. Alveolar macrophages (AMs) are essential for maintaining lung homeostasis and play a significant role in the development of lung fibrosis. Tissue-Resident Alveolar Macrophages (TR-AMs), which originate from embryonic progenitors, can self-renew locally in a steady state, independent of hematopoiesis. During fibrogenesis, circulating monocytes rapidly migrate into the lungs and differentiate into monocyte-derived AMs (Mo-AMs). MicroRNAs (miRNAs), small non-coding RNAs, are critical for regulating gene expression. Our recent study found that the loss of miRNAs in embryonic progenitors significantly decreased the number of TR-AMs in late-stage embryos, indicating that miRNAs are necessary for TR-AM development. However, the role of miRNAs in the postnatal maintenance of TR-AMs and Mo-AMs, as well as their function in pulmonary fibrosis, remains unclear. Methods and Results:Here, we demonstrate that deleting miRNAs after birth severely disrupts TR-AM homeostasis and Mo-AM repopulation from the bone marrow following irradiation. The deficiency of miRNAs in TR-AMs and Mo-AMs was linked to diminished bleomycin-induced experimental lung fibrosis. Mechanistically, the absence of miRNAs increased TR-AM apoptosis under both normal and fibrotic conditions. RNA sequencing (RNA-seq) analysis revealed distinct transcriptomic and pathway changes in miRNA-deficient AM subgroups after lung injury. The integration of RNA-seq and miRNA array analyses identified miRNA-mRNA networks in TR-AMs and Mo-AMs in response to bleomycin injury. Ingenuity Pathway Analysis further predicted let-7a, miR-155, and miR-125 as unique upstream regulators of Mo-AM responses to lung fibrosis. Conclusions:Our findings suggest that miRNAs are key epigenetic mediators that differentially regulate the maintenance and function of TR-AMs and Mo-AMs in the pathogenesis of pulmonary fibrosis.

Invariant natural killer T (iNKT) cells are innate-like T cells that are abundant in liver sinusoids and play a critical role in tumor immunity. However, the role of iNKT cells in pancreatic cancer liver metastasis (PCLM) has not been fully explored. In this study, we employed a hemi-spleen pancreatic tumor cell injection mouse model of PCLM, a model that closely mimics clinical conditions in humans, to explore the role of iNKT cells in PCLM. Activation of iNKT cells with α-galactosylceramide (αGC) markedly increased immune cell infiltration and suppressed PCLM progression. Via single cell RNA sequencing (scRNA-seq) we profiled over 30,000 immune cells from normal liver and PCLM with or without αGC treatment and were able to characterize the global changes of the immune cells in the tumor microenvironment upon αGC treatment, identifying a total of 12 subpopulations. Upon treatment with αGC, scRNA-Seq and flow cytometry analyses revealed increased cytotoxic activity of iNKT/NK cells and skewing CD4 T cells towards a cytotoxic Th1 profile and CD8 T cells towards a cytotoxic profile, characterized by higher proliferation and reduced exhaustion marker PD1 expression. Moreover, αGC treatment excluded tumor associated macrophages. Lastly, imaging mass cytometry analysis uncovered the reduced epithelial to mesenchymal transition related markers and increased active CD4 and CD8 T cells in PCLM with αGC treatment. Overall, our findings uncover the protective function of activated iNKT cells in pancreatic cancer liver metastasis through increased NK and T cell immunity and decreased tumor associated macrophages.

Abstract Epidermal Langerhans cells (LCs) derive from embryonic myeloid progenitors at the steady-state and monocyte progenitors under inflammatory conditions. LCs have the capacity to induce both immunity and tolerance in the skin, but how a single population of LCs mediates both these functions has perplexed researchers for decades. We hypothesized that LCs in murine epidermis have functionally heterogenous subpopulations. We employed single-cell RNA sequencing (scRNAseq) and scATACseq to identify transcriptional and epigenetic heterogeneity in LCs during late embryonic development, adult steady-state and inflamed-state. We found three transcriptionally distinct clusters in adult at steady-state: ATF3hi CD207lo (LC1), ATF3lo CD207hi(LC2), and CD207+ cells expressing keratinocyte (KC) genes (kLCs). Ingenuity pathway analysis showed LC1 had downregulated immunostimulatory pathways and LC2 had upregulated immunostimulatory pathways. LCs from ATF3 knockout mice promoted Th1/2/17 immunity in co-culture experiments, confirming the immunotolerant function of LC1s. LC1 and LC2 clusters had corresponding scATACseq clusters but kLCs did not, suggesting that kLCs may acquire cellular material (i.e. mRNA) through interactions with KCs. scRNAseq analyses of E18.5 pre-LCs and 3 weeks post UVC-treatment also identified ATF3hi and ATF3lo clusters, but kLCs were neither present at E18.5 nor after UVC treatment. Overall, our single cell analyses uncover murine epidermal LC subsets with distinct functions during late embryonic development, steady-state and inflamed-state. Supported by NIH/NIAMS, R01AR078688-01A1

Angiotensin-converting enzyme 2 (ACE2) receptor is required for SARS-CoV-2 entry into human cells. However, emerging evidence shows SARS-CoV-2 infected lung monocytes/macrophages from COVID-19 patients barely express ACE2 mRNA, raising a question how SARS-CoV-2 penetrates macrophages. It’s also under debating whether the peripheral blood cells (HPBCs) can be infected by SARS-CoV-2 that may facilitate viral spread from circulation to other organs besides lung. Herein we demonstrate that resting primary HPBCs harbor abundant cytoplasmic ACE2, regardless of COVID-19 status, and that surface translocation is necessary for viral infection. Upon ex vivo TLR4/7/8 stimulation of HPBCs, ACE2 translocated to the cell surface independent of ACE2 transcription, and this translocation was blocked by an endosomal trafficking inhibitor, suggesting the putative source as ACE2-containing exosomes. However, only stimulated monocytes concurrently expressing ACE2 and cell surface transmembrane serine protease type 2 (TMPRSS2) were efficiently infected by SARS-CoV-2, which was significantly mitigated by remdesivir. Furthermore, ACE2 surface translocation in peripheral myeloid cells from patients with severe COVID-19 correlated with their proinflammatory cytokine production. Collectively, TLR4/7/8-induced ACE2 translocation with TMPRSS2 expression is indispensable for SARS-CoV-2 infection of circulating monocytes. Our work not only provides a new mechanism for the pathogenesis of SARS-CoV-2 and a potential path for its systemic infection, but also unveils a prospective therapeutic strategy by targeting ACE2 trafficking for preventing monocyte/macrophage infection. This study is partially supported by National Institutes of Health grants R61AR076803, R01AR063611, R01AI119041, and R01AR069681 (Q-S. M.), R01AR072046 (L.Z.), R01DK120623 (J.Z.S.), Henry Ford Immunology Program grants (T71016, Q-S. M.; T71017, L. Z.), and funding for the U-M Center for Drug Repurposing - NCATS CTSA UL1TR002240 (J.Z.S.).

Infection of human peripheral blood cells by SARS-CoV-2 has been debated because immune cells lack mRNA expression of both angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease type 2 (TMPRSS2). Herein we demonstrate that resting primary monocytes harbor abundant cytoplasmic ACE2 and TMPRSS2 protein and that circulating exosomes contain significant ACE2 protein. Upon ex vivo TLR4/7/8 stimulation, cytoplasmic ACE2 was quickly translocated to the monocyte cell surface independently of ACE2 transcription, while TMPRSS2 surface translocation occurred in conjunction with elevated mRNA expression. The rapid translocation of ACE2 to the monocyte cell surface was blocked by the endosomal trafficking inhibitor endosidin 2, suggesting that endosomal ACE2 could be derived from circulating ACE2-containing exosomes. TLR-stimulated monocytes concurrently expressing ACE2 and TMPRSS2 on the cell surface were efficiently infected by SARS-CoV-2, which was significantly mitigated by remdesivir, TMPRSS2 inhibitor camostat, and anti-ACE2 antibody. Mass cytometry showed that ACE2 surface translocation in peripheral myeloid cells from patients with severe COVID-19 correlated with its hyperactivation and PD-L1 expression. Collectively, TLR4/7/8-induced ACE2 translocation with TMPRSS2 expression makes circulating monocytes permissive to SARS-CoV-2 infection.

As the initiators of adaptive immune responses, DCs play a central role in regulating the balance between CD8 T cell immunity versus tolerance to tumor antigens. Exploiting their function to potentiate host anti-tumor immunity, DC-based vaccines have been one of most promising and widely used cancer immunotherapies. However, DC-based cancer vaccines have not achieved the promised success in clinical trials, with one of the major obstacles being tumor-mediated immunosuppression. A recent discovery on the critical role of type 1 conventional DCs (cDC1s) play in cross-priming tumor-specific CD8 T cells and determining the anti-tumor efficacy of cancer immunotherapies, however, has highlighted the need to further develop and refine DC-based vaccines either as monotherapies or in combination with other therapies. DC-derived exosomes (DCexos) have been heralded as a promising alternative to DC-based vaccines, as DCexos are more resistance to tumor-mediated suppression and DCexo vaccines have exhibited better anti-tumor efficacy in pre-clinical animal models. However, DCexo vaccines have only achieved limited clinical efficacy and failed to induce tumor-specific T cell responses in clinical trials. The lack of clinical efficacy might be partly due to the fact that all current clinical trials used peptide-loaded DCexos from monocyte-derived DCs. In this review, we will focus on the perspective of expanding current DCexo research to move DCexo cancer vaccines forward clinically to realize their potential in cancer immunotherapy.

Traditional immunohistochemistry (IHC) is inherently limited by its ability to analyze only several markers within a histological tissue section at a given time, which hinders in-depth characterization and phenotyping of tissues. Imaging mass cytometry (IMC), which combines IHC using metal-labeled antibodies with laser ablation and detection using mass cytometry by time-of-flight, overcomes this limitation with the capability to simultaneously analyze up to 40 protein markers to generate high-dimensional images from a single tissue section. IMC analysis preserves tissue architecture and spatial cellular relationships that would otherwise be lost or significantly altered in applications requiring tissue dissociation, such as flow cytometry or single-cell RNA sequencing. Resulting high-dimensional histological images permit spatially conserved analysis to identify unique cell populations, cellular interactions and avoidances, and insight into activation and behavioral status based on tissue location. IMC can be performed on both frozen and formalin-fixed paraffin-embedded tissue, allowing for previously banked samples to be analyzed and correlated with known clinical outcomes. Expectedly, IMC will change the landscape of investigative pathology, particularly when used in coordination with multiomic platforms to combine transcriptomic and proteomic data at a single-cell resolution. Here, we aim to highlight the potential utility of IMC within dermatologic research and clinical applications.

Mouse epidermis contains two major immune cells, antigen presenting Langerhans cells (LCs) and dendritic epidermal T cells (DETCs), which regulate skin immunity and involve in disease pathogenesis. Recent lineage-tracing studies have uncovered that mouse DETCs and LCs are derived from embryonic yolk-sac-derived hematopoietic precursors and self-maintain after birth. However, detailed regulating mechanisms related to their ontogeny and homeostasis remain unclear. MicroRNAs (miRNAs) are important post transcriptional regulators of protein-coding genes. Using a CSF1R-cre-mediated Dicer deletion mouse model, we found the critical involvement of miRNAs in the ontogeny of both LCs and DETCs. To further investigate the role of individual miRNAs in the ontogeny of LCs and DETCs, we evaluated the expression of miRNAs in the precursors of LCs and DETCs at different embryonic developmental stages and identified that miR17-92 cluster was highly expressed and dynamically regulated. We next generated a Csf1rCre-mediated miR17-92 deletion mouse model (miR17-92 KO) and found that conditionally deletion of miR17-92 led to significantly reduced number and interrupted phenotypes of epidermis LC and DETC precursors at E18.5 and P0 of miR17-92 KO embryos. Defective LC precursors were also identified at E14.5 and E16.5 embryonic skin, while diminished number and blocked maturation of Vg3+ DETC precursors were identified in the thymus of the same embryonic stages in miR17-92 KO mice. Overall, our results indicate that miR17-92 serves as a key epigenetic regulator in the ontogeny of LCs and DETCs. Detailed molecular mechanisms underlining the miR17-92 mediated LC and DETC developmental regulation are currently under investigation.

Alveolar macrophages (AMs) derived from embryonic precursors seed the lung before birth and self-maintain locally throughout adulthood, but are regenerated by bone marrow (BM) under stress conditions. However, the regulation of AM development and maintenance remains poorly understood. Here, we show that histone deacetylase 3 (HDAC3) is a key epigenetic factor required for AM embryonic development, postnatal homeostasis, maturation, and regeneration from BM. Loss of HDAC3 in early embryonic development affects AM development starting at E14.5, while loss of HDAC3 after birth affects AM homeostasis and maturation. Single-cell RNA sequencing analyses reveal four distinct AM sub-clusters and a dysregulated cluster-specific pathway in the HDAC3-deficient AMs. Moreover, HDAC3-deficient AMs exhibit severe mitochondrial oxidative dysfunction and deteriorative cell death. Mechanistically, HDAC3 directly binds to Pparg enhancers, and HDAC3 deficiency impairs Pparg expression and its signaling pathway. Our findings identify HDAC3 as a key epigenetic regulator of lung AM development and homeostasis.

Alveolar macrophages (AMs) are derived from embryonic precursors that seed the lung before birth and self-maintain locally throughout adulthood but can be regenerated by bone marrow (BM) under stress conditions. However, how to precisely regulate AM development still remains unknown. Here we report that histone deacetylase 3 (HDAC3) is a key epigenetic factor required for AM embryonic development, postnatal homeostasis, maturation, and regeneration from BM. Loss of HDAC3 in early embryonic development affects AM development starting at E14.5, while loss of HDAC3 after birth affects AM homeostasis and maturation. HDAC3-deficient AMs exhibit severe mitochondrial oxidative dysfunction and deteriorative cell death. Single-cell RNA sequencing analyses further identified four distinct AM sub-clusters and a lack of HDAC3 dysregulated AM transcriptome in a cluster-specific manner. Mechanistically, HDAC3 serves as a co-activator via deacetylation of PU.1 to promote Pparg transcription. Our findings identify HDAC3 as a key epigenetic regulator of lung AM development and homeostasis.

Epidermal Langerhans cells (LCs), skin-residing dendritic cells (DCs), control both induction of adaptive immunity and immune tolerance in skin and are involved in the development of skin diseases, including skin allergy and atopic dermatitis. Recent lineage-tracing studies have uncovered that rather than from bone marrow (BM)-derived DC precursors, adult mouse LCs are derived from embryonic yolk sac–derived macrophages and fetal liver monocytes.1Ginhoux F. Schultze J.L. Murray P.J. Ochando J. Biswas S.K. New insights into the multidimensional concept of macrophage ontogeny, activation and function.Nat Immunol. 2016; 17: 34-40Crossref PubMed Scopus (465) Google Scholar Like epidermal LCs, tissue-resident macrophages (TRMs) also follow unique patterns of ontogeny, deriving from embryonic yolk sac–derived macrophages or fetal liver monocytes, and they play critical roles in tissue development, homeostasis, and the development of some diseases. In contrast to the infiltrating monocytes, which are recruited via extravasation from blood vessels after local inflammation initiation and then produce inflammatory mediators but do not persist after the resolution of inflammation, the TRMs proliferate locally and are involved in the inflammation initiation or inducing tolerance, and then back to the quiescence following remission, and persist locally (see additional references in this article's Online Repository at www.jacionline.org). TRMs in the skin and lungs have been shown to have an impact on the progression and resolution of allergic inflammation, such as allergic contact dermatitis2Kaplan D.H. Igyarto B.Z. Gaspari A.A. Early immune events in the induction of allergic contact dermatitis.Nat Rev Immunol. 2012; 12: 114-124Crossref PubMed Scopus (383) Google Scholar and asthma.3Draijer C. Peters-Golden M. Alveolar macrophages in allergic asthma: the forgotten cell awakes.Curr Allergy Asthma Rep. 2017; 17: 12Crossref PubMed Scopus (48) Google Scholar Up to now, a few genes have been identified to regulate LC and TRM embryonic development, including cytokines IL-34 and GM-CSF, which are required for the development of LCs and lung-resident alveolar macrophages (AMs), respectively, as well as TGF-β, which is essential for both LC and lung AM ontogeny and homeostasis4Zhang X. Gu J. Yu F.S. Zhou L. Mi Q.S. TGF-beta1-induced transcription factor networks in Langerhans cell development and maintenance.Allergy. 2016; 71: 758-764Crossref PubMed Scopus (16) Google Scholar (see additional references in this article's Online Repository). MicroRNAs (miRNAs), a class of evolutionarily conserved small noncoding RNAs, negatively regulate the expression of protein-coding genes and are involved in immune cell development and function.5Zhou L. Seo K.H. He H.Z. Pacholczyk R. Meng D.M. Li C.G. et al.Tie2cre-induced inactivation of the miRNA-processing enzyme Dicer disrupts invariant NKT cell development.Proc Natl Acad Sci U S A. 2009; 106: 10266-10271Crossref PubMed Scopus (68) Google Scholar However, it is totally unknown whether miRNAs are involved in LC and TRM ontogeny. The RNase III enzyme DICER is essential for the processing of mature and functional miRNAs; therefore, its deletion provides a genetic test for miRNA function. The conditional inactivation of DICER in the BM and the thymus has indicated the requirement for miRNA activity in regulatory T-cell and natural killer T-cell development.5Zhou L. Seo K.H. He H.Z. Pacholczyk R. Meng D.M. Li C.G. et al.Tie2cre-induced inactivation of the miRNA-processing enzyme Dicer disrupts invariant NKT cell development.Proc Natl Acad Sci U S A. 2009; 106: 10266-10271Crossref PubMed Scopus (68) Google Scholar Interestingly, deletion of DICER in BM myeloid lineage by LysMCre does not affect monocyte and macrophage development or even tumor-associated macrophages.6Baer C. Squadrito M.L. Laoui D. Thompson D. Hansen S.K. Kiialainen A. et al.Suppression of microRNA activity amplifies IFN-gamma-induced macrophage activation and promotes anti-tumour immunity.Nat Cell Biol. 2016; 18: 790-802Crossref PubMed Scopus (179) Google Scholar Recently, colony-stimulating factor 1 receptor (CSF1R) Cre reporter (called Csf1rCre) was used to fate map embryonic TRM and LC precursor development.7Schulz C. Gomez Perdiguero E. Chorro L. Szabo-Rogers H. Cagnard N. Kierdorf K. et al.A lineage of myeloid cells independent of Myb and hematopoietic stem cells.Science. 2012; 336: 86-90Crossref PubMed Scopus (1724) Google Scholar To study the role of miRNAs in the embryonic development of LCs and TRMs, we crossed Csf1rCre mice with Dicerfl/fl mice to generate Csf1rCreDicerfl/fl conditional knockout (cKO) mice, in which DICER is deficient in the CSF1R-expressing cells including LCs, TRMs, and monocytes.7Schulz C. Gomez Perdiguero E. Chorro L. Szabo-Rogers H. Cagnard N. Kierdorf K. et al.A lineage of myeloid cells independent of Myb and hematopoietic stem cells.Science. 2012; 336: 86-90Crossref PubMed Scopus (1724) Google Scholar As expected, the expression of all tested miRNAs in BM-derived macrophages (gating strategy shown in Fig E1, A, in this article's Online Repository at www.jacionline.org) from DICER cKO mice was completely diminished or dramatically reduced (Fig 1, A). We first compared the frequencies of epidermal LCs and TRMs in the different tissues between wild-type (WT) Dicerfl/fl and mutant Csf1rCreDicerfl/fl adult mice (gates shown in Fig E1, B). A previous study reported that the number of epidermal LCs, not DCs, were dramatically reduced in DC lineage CD11cCre-induced DICER deletion mice.8Kuipers H. Schnorfeil F.M. Fehling H.J. Bartels H. Brocker T. Dicer-dependent microRNAs control maturation, function, and maintenance of Langerhans cells in vivo.J Immunol. 2010; 185: 400-409Crossref PubMed Scopus (64) Google Scholar However, the role of miRNAs in epidermal LC ontogeny remains unknown. Consistent with this report, we found that CD45+MHC II+ epidermal LCs were almost completely diminished in the DICER cKO mice at 10 weeks old (Fig 1, B). Surprisingly, unlike the previous report on the deletion of DICER in BM myeloid lineage,6Baer C. Squadrito M.L. Laoui D. Thompson D. Hansen S.K. Kiialainen A. et al.Suppression of microRNA activity amplifies IFN-gamma-induced macrophage activation and promotes anti-tumour immunity.Nat Cell Biol. 2016; 18: 790-802Crossref PubMed Scopus (179) Google Scholar F4/80brightCD11blo dermal-resident macrophages, CD11c+SiglecF+ lung-resident AMs, and F4/80brightCD11blo kidney-resident macrophages were almost completely diminished in the DICER cKO mice, whereas other TRMs including F4/80brightCD11blo brain-resident macrophage (microglia) and liver-resident macrophages (Kupffer cells) remained constant (Fig 1, B). The reduction of miRNA expression was further confirmed in the TRMs of lung and brain from DICER-deficient mice (Fig 1, C). These results suggest that miRNAs are likely involved in the ontogeny and/or homeostasis of skin LCs and TRMs in the skin, lungs, and kidneys. Next, we asked whether miRNAs are required for the embryonic development of LCs and TRMs. We compared the frequencies of TRMs in the skin, lungs, kidneys, brain, and liver between WT and DICER-deficient embryos at gestation day 16.5 (E16.5), E18.5, and postnatal day 0 (P0) (gates shown in Fig E1, C). As shown in Fig 2, the frequencies of skin-resident macrophages remained unaltered in DICER-deficient embryos compared with their WT counterparts at E16.5. Given that the mouse epidermis is fully mature after E17.5, we further analyzed the epidermis-LC precursors and dermis-resident macrophages, respectively, at E18.5 and P0. We found that the frequencies of epidermis-LC precursors and dermis-resident macrophages were remarkably decreased in Csf1rCreDicerfl/fl cKO embryos at both E18.5 and P0. Likewise, although the frequencies of lung-resident macrophages were comparable between WT and DICER cKO embryos at E16.5, we observed a dramatic reduction in the frequencies of lung-resident AM in DICER cKO embryos at E18.5 and P0 (Fig 2). Thus, miRNA deficiency leads to impaired LC, skin- and lung-resident macrophage development at late embryonic stage. Furthermore, the frequencies of F4/80brightCD11blo TRMs in the kidneys were significantly decreased at P0 in the DICER-deficient pups, although the frequencies remained unchanged between WT and Dicer-deficient embryos at E16.5 and E18.5 (Fig 2). Thus, we reason that miRNAs may be dispensable for embryonic development of kidney-resident macrophages, but are likely required for TRM migration to and seeding to the kidneys, or for maintenance of TRMs in the kidneys, which could contribute to the decreased frequency of F4/80brightCD11blo cells in the kidneys from adult DICER cKO mice (Fig 1, B). Interestingly, the frequencies of TRMs in the brain and liver were comparable in the DICER cKO embryos/newborns at E16.5, E18.5, and P0 (Fig 2), compared with WT embryos. Taken together, our data suggest that miRNAs may not be required for the embryonic development of TRMs in the kidneys, brain, and liver. However, given that very few miRNAs are DICER-independent,9Kim Y.K. Kim B. Kim V.N. Re-evaluation of the roles of DROSHA, Export in 5, and DICER in microRNA biogenesis.Proc Natl Acad Sci U S A. 2016; 113: E1881-E1889Crossref PubMed Scopus (275) Google Scholar it remains a possibility that those DICER-independent miRNAs may contribute to TRM ontogeny in the kidneys, brain, and liver. In summary, our data suggest that miRNAs serve as critical epigenetic regulators in the ontogeny of LCs as well as TRMs in the skin and lungs. Although the specific miRNA(s) required for LC and TRM ontogeny remain to be determined, our work provides a new hint to study the miRNA regulation of LCs and TRMs. Uncovering novel LC and TRM-associated miRNAs may potentially help in the development of specific miRNA therapies for some allergic diseases in the skin and lungs. We thank all laboratory members for their help and encouragement. Csf1rCre (Strain #021024)E1Deng L. Zhou J.F. Sellers R.S. Li J.F. Nguyen A.V. Wang Y. et al.A novel mouse model of inflammatory bowel disease links mammalian target of rapamycin-dependent hyperproliferation of colonic epithelium to inflammation-associated tumorigenesis.Am J Pathol. 2010; 176: 952-967Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar and Dicerfl/fl (Strain #006001)E2Zhou L. Seo K.H. He H.Z. Pacholczyk R. Meng D.M. Li C.G. et al.Tie2cre-induced inactivation of the miRNA-processing enzyme Dicer disrupts invariant NKT cell development.Proc Natl Acad Sci U S A. 2009; 106: 10266-10271Crossref PubMed Scopus (81) Google Scholar were purchased from Jackson Laboratories (Bar Harbor, Me). All animal experiments were approved by the Institutional Animal Care and Use Committee of Henry Ford Health System and performed in accordance with the National Institutes of Health guidelines. Whole tissues from embryos, newborns, or adult mice were homogenized by pipetting or smashing with slides, incubated at 37°C in a shaking incubator for 30 minutes in PBS containing 3% FBS (Hyclone, Pittsburgh, Pa), 1 mg/mL collagenase D (Roche Diagnostics, South San Francisco, Calif), and 100 U/mL DNase I (Worthington, Lakewood, NJ), and then passed through a 40-μm nylon mesh (Thermo Fisher Scientific, Pittsburgh, Pa). Red blood cells were lysed by incubation with 1× red blood cell lysis buffer (0.15 mol NH4Cl, 10 mM NaHCO3, and 0.1 mM EDTA) at room temperature for 4 minutes. Whole skins from E18.5 embryos, newborns, and adult mice were incubated at 37°C for 1 hour in PBS containing 0.25% Dispase (Thermo Fisher Scientific). The epidermal layer was detached and further incubated in a 37°C water bath with shaking for 15 minutes in PBS containing 0.05% trypsin (Corning Cellgro, Pittsburgh, Pa) and 100 U/mL DNase I. The epidermal samples were passed through a 40-μm nylon mesh to obtain single-cell suspensions. The samples prepared from skin and other tissues were incubated on ice for 15 minutes with anti-CD16/CD32 mAb (Tonbo Biosciences, clone 2.4G2, San Diego, Calif) for Fc blocking, and stained on ice for 30 minutes with a mixture of fluorescent antibodies including MHC II-FITC (I-A/I-E, eBioscience, clone M5/114.15.2, Pittsburgh, Pa), CD11b-PerCP-Cy5.5 (BioLegend, clone M1/70, San Diego, Calif), CD45-APC-eFluor 780 (eBioscience, clone 30-F11), F4/80-efluor 450 (eBioscience, clone BM8), Siglec F-PE (BD Biosciences, clone E50-2440, San Jose, Calif), and CD11c-APC (eBioscience, clone N418). The BM-derived macrophages were isolated according to a standard protocol as described previously.E3Zhang X. Goncalves R. Mosser D.M. The isolation and characterization of murine macrophages.Curr Protoc Immunol. 2008; (Chapter 14:Unit 14 1)Crossref Scopus (1083) Google Scholar The samples were acquired or sorted with a Becton Dickinson FACSAria II flow cytometer (BD Biosciences), and the data were analyzed by the FlowJo software (Tree Star, Ashland, Ore). Total RNA was extracted from sorted BM-derived macrophages, lung AM, and brain microglia of WT or Csf1rCreDicerfl/fl knockout mice with miRNeasy Mini Kit (QIAGEN, Gaithersburg, Md). The RNA was reverse-transcribed to cDNA with miRCURY LNA microRNA RT Kit (Exiqon/QIAGEN). Quantitative real-time PCR reactions were prepared using FastStart Universal SYBR Green Master (ROX, Roche) and carried out in QuantStudio 7 Flex Real-Time PCR System (Applied Biosystems, Foster City, Calif). The U6 primer set was purchased from Exiqon (Product No. 203907). All other miRNA primer sets were purchased from Thermo Fisher Scientific, including miR-17 (Assay ID 002308), miR-18a (Assay ID 002422), miR-19a (Assay ID 000395), miR-19b (Assay ID 000396), miR-20a (Assay ID 000580), miR-92a (Assay ID 000430), miR-21 (Assay ID 000397), miR-146a (Assay ID 000468), miR-150 (Assay ID 000473), miR-155 (Assay ID 002571), and miR-233 (Assay ID: 002295). Relative miRNA expressions were normalized to U6 expression. All data were collected from at least 2 independent experiments. For all continuous data, if the data were nonnormal by the Shapiro-Wilk test, a nonparametric Mann-Whitney U test was used. For normal distributed data, if variances were equal, Student t test was used; otherwise, unpaired t test with Welch correction is used. Statistical analyses were performed with Prism 7 software (GraphPad, La Jolla, Calif).

The epidermis-resident γδ T cells (termed dendritic epidermal T cells [DETCs]) are the first T cells that develop during thymic ontogeny. Their progenitors, Vγ3+Vδ1+ (Garman nomenclature) cells, appear in the fetal thymus at gestation day 14.5 (E14.5), expand significantly before emerging from the thymus at E16.5, home to the epidermis at E18.5, and then self-maintain during their whole life (Macleod and Havran, 2011). Although many advances in our understanding of γδ T cells have been made over the last decade, lack of a valid embryonic fate mapping tool significantly blocks the in-depth investigation of resident γδ T-cell embryonic development.

Mass cytometry or CyTOF is an emerging technology for high-dimensional multiparameter single cell analysis that overcomes many limitations of fluorescence-based flow cytometry. New methods for analyzing CyTOF data attempt to improve automation, scalability, performance, and interpretation of data generated in large studies. Assigning individual cells into discrete groups of cell types (gating) involves time-consuming sequential manual steps, untenable for larger studies. We introduce DeepCyTOF, a standardization approach for gating, based on deep learning techniques. DeepCyTOF requires labeled cells from only a single sample. It is based on domain adaptation principles and is a generalization of previous work that allows us to calibrate between a target distribution and a source distribution in an unsupervised manner. We show that Deep-CyTOF is highly concordant (98%) with cell classification obtained by individual manual gating of each sample when applied to a collection of 16 biological replicates of primary immune blood cells, even when measured accross several instruments. Further, DeepCyTOF achieves very high accuracy on the semi-automated gating challenge of the FlowCAP-I competition as well as two CyTOF datasets generated from primary immune blood cells: (i)14 subjects with a history of infection with West Nile virus (WNV), (ii) 34 healthy subjects of different ages. We conclude that deep learning in general, and DeepCyTOF specifically, offers a powerful computational approach for semi-automated gating of CyTOF and flow cytometry data.

West Nile virus (WNV) infection is mainly asymptomatic but can be severe in elderly persons. As part of studies on immunity and aging in Connecticut, USA, we detected WNV seroconversion in 8.5% of nonimmunosuppressed and 16.8% of immunosuppressed persons. Age was not a significant seroconversion factor. Our findings suggest that immune factors affect seroconversion.

Langerhans cells (LCs) are epidermal-resident dendritic cells that play important roles in skin immunity and tolerance. Recent fate-mapping studies have demonstrated that adult LCs derive predominantly from fetal liver monocytes with a minor contribution of yolk sac-derived macrophages. However, the role of epigenetic regulations in embryonic development of LCs remains unclear. MicroRNAs (miRNAs) are small, noncoding RNAs that negatively regulate gene expression of their mRNA targets. We and others recently reported that miRNAs are key players in regulating immune cell development and function, including LCs. To further study the role of miRNAs in LC ontogeny, we generated CSF1rCreDicerf/fKO mice in which miRNAs are deficient in myeloid lineage cells including LCs. As expected, the frequency of LCs was drastically reduced in Dicer KO adult mice. Interestingly, there was also a significant reduction (p=0.027) of epidermal LCs in Dicer KO embryos at E18.5, suggesting that miRNAs are crucial for embryonic development of LCs. Given the differential expression of miR-150 and miR-155 in embryonic LC development, we generated CSF1rCremiR-150f/fKI and CSF1rCremiR-155f/fKI mice in which expression of miR-150 and miR-155 is upregulated in LC-lineage, respectively. Surprisingly, neither strain caused defective LC development in fetal or adult epidermis. Overall, this is the first study to demonstrate that miRNAs are required for embryonic LC development. Furthermore, although overexpression of miR-150 or miR-155 is dispensable for LC ontogeny, the specific miRNAs responsible for proper LC development deserve to be further explored.

Immunodeficient mice reconstituted with a human immune system represent a promising tool for translational research as they may allow modeling and therapy of human diseases in vivo. However, insufficient development and function of human natural killer (NK) cells and T cell subsets limit the applicability of humanized mice for studying cancer biology and therapy. Here, we describe a human interleukin 15 (IL15) and human signal regulatory protein alpha (SIRPA) knock-in mouse on a Rag2-/- Il2rg-/- background (SRG-15). Transplantation of human hematopoietic stem and progenitor cells into SRG-15 mice dramatically improved the development and functional maturation of circulating and tissue-resident human NK and CD8+ T cells and promoted the development of tissue-resident innate lymphoid cell (ILC) subsets. Profiling of human NK cell subsets by mass cytometry revealed a highly similar expression pattern of killer inhibitory receptors and other candidate molecules in NK cell subpopulations between SRG-15 mice and humans. In contrast to nonobese diabetic severe combined immunodeficient Il2rg-/- (NSG) mice, human NK cells in SRG-15 mice did not require preactivation but infiltrated a Burkitt's lymphoma xenograft and efficiently inhibited tumor growth following treatment with the therapeutic antibody rituximab. Our humanized mouse model may thus be useful for preclinical testing of novel human NK cell-targeted and combinatory cancer immunotherapies and for studying how they elicit human antitumor immune responses in vivo.

Abstract Microglia are tissue resident macrophages of the central nervous system (CNS) that function in CNS maintenance and innate immune defense. Macrophages are mediators of immune threshold and thus implicated in disease pathogenesis. Compared to research efforts on CNS microglia, relatively little is known about microglial development or homeostasis in the retina. Histone deacetylases (HDACs) are enzymes known to regulate gene expression by modifying chromatin structure. Here, we use a murine model with conditional deletion of HDAC3 induced by Colony Stimulating Factor 1 Receptor (Csf1r) Cre to determine the consequence of HDAC3 depletion on microglial development and homeostasis in the retina. Macrophages were identified using CD11b and F4/80 markers. Retinal microglial populations were assessed in mice at embryonic day 14.5 (E14.5) as well as at birth and 4-weeks old. A population of CD11b+F4/80bright retinal microglia were identified as early as E14.5, indicating that retinal microglia are derived from seeding of embryonic progenitors from the yolk sac or fetal liver. Although the frequency of retinal microglia was comparable at embryonic stage and at birth, it was significantly reduced (P = 0.008) in HDAC3 KO mice at 4 weeks old. Moreover, retinal microglia were partially radio-resistant, and could be repopulated by fetal liver cells transferred from WT but not HDAC3 KO embryos. This is the first report to show that retina-resident microglia are derived from embryonic precursors and that HDAC3 controls microglial homeostasis in adult retina. HDAC3 may serve as a promising therapeutic target for microglial immune-reactive pathologies such as age-related macular degeneration.

OBJECTIVES:The aim of this study was to elucidate the molecular mechanisms by which food-derived casein glycomacropeptide (CGMP) maintains internal homeostasis in the intestinal mucosa and to investigate the effects of CGMP on the intestinal mucosal immunological barrier and related signal transduction pathways. METHODS:In this study, a famoxadone (OXZ)-induced mouse experimental ulcerative colitis (UC) model was built. The experimental UC mice were intragastrically administered milk-derived CGMP for four consecutive days. The molecular mechanisms by which milk-derived CGMP improved and restored the inflammatory status in UC symptoms were elucidated by H&E staining, immunohistochemical staining and western blotting. RESULTS:The results indicated that CGMP (50 mg/(kg bw·d)) could significantly improve morphological injury to intestinal mucosa in OXZ-induced UC mice to the same extent that did sulfasalazine (SASP, 40 mg/(kg bw·d)), a medicine used to treat UC, in the control group. The study found that CGMP could significantly reduce the expression of Human mucosal addressin cell adhesion molecule-1 (MAdCAM-1), Cluster of differentiation 4 (CD4) and Cluster of differentiation 8 (CD8) in the lamina propria of the intestinal mucosa and significantly stimulate the secretion of sIgA to increase intestinal immunity. Furthermore, CGMP was found to be directly involved in inhibiting the MAPK pathway and activating the TGF-β1/Smad signal transduction cascade, which could maintain immunological regulation of the intestinal mucosa and protect the functions of the intestinal mucosal barrier. CONCLUSIONS:This study elucidated the molecular mechanisms by which CGMP maintained homeostasis of the intestinal mucosa and further confirmed its pharmaceutical value as a food-derived functional component with promising potential for further exploration/utilization.