Gpr75 knockout mice display age-dependent cone photoreceptor cell loss.

GPR75 is an orphan G protein-coupled receptor for which there is currently limited information and its function in physiology and disease is only recently beginning to emerge. This orphan receptor is expressed in the retina but its function in the eye is unknown. The earliest studies on GPR75 were conducted in the retina, where the receptor was first identified and cloned and mutations in the receptor were identified as a possible contributor to retinal degenerative disease. Despite these sporadic reports, the function of GPR75 in the retina and in retinal disease has yet to be explored. To assess whether GPR75 has a functional role in the retina, the retina of Gpr75 knockout mice was characterized. Knockout mice displayed a mild progressive retinal degeneration, which was accompanied by oxidative stress. The degeneration was because of the loss of both M-cone and S-cone photoreceptor cells. Housing mice under constant dark conditions reduced oxidative stress but did not prevent cone photoreceptor cell loss, indicating that oxidative stress is not a primary cause of the observed retinal degeneration. Studies here demonstrate an important role for GPR75 in maintaining the health of cone photoreceptor cells and that Gpr75 knockout mice can be used as a model to study cone photoreceptor cell loss.

Acyl-CoA:wax alcohol acyltransferase 2 modulates the cone visual cycle in mouse retina.

The daylight and color vision of diurnal vertebrates depends on cone photoreceptors. The capability of cones to operate and respond to changes in light brightness even under high illumination is attributed to their fast rate of recovery to the ground photosensitive state. This process requires the rapid replenishing of photoisomerized visual chromophore (11-cis-retinal) to regenerate cone visual pigments. Recently, several gene candidates have been proposed to contribute to the cone-specific retinoid metabolism, including acyl-CoA wax alcohol acyltransferase 2 (AWAT2, aka MFAT). Here, we evaluated the role of AWAT2 in the regeneration of visual chromophore by the phenotypic characterization of Awat2-/- mice. The global absence of AWAT2 enzymatic activity did not affect gross retinal morphology or the rate of visual chromophore regeneration by the canonical RPE65-dependent visual cycle. Analysis of Awat2 expression indicated the presence of the enzyme throughout the murine retina, including the retinal pigment epithelium (RPE) and Müller cells. Electrophysiological recordings revealed reduced maximal rod and cone dark-adapted responses in AWAT2-deficient mice compared to control mice. While rod dark adaptation was not affected by the lack of AWAT2, M-cone dark adaptation both in isolated retina and in vivo was significantly suppressed. Altogether, these results indicate that while AWAT2 is not required for the normal operation of the canonical visual cycle, it is a functional component of the cone-specific visual chromophore regenerative pathway.

Retinal degeneration in mice expressing the constitutively active G90D rhodopsin mutant.

Rhodopsin is the G protein-coupled receptor in rod photoreceptor cells that initiates vision upon photon capture. The light receptor is normally locked in an inactive state in the dark by the covalently bound inverse agonist 11-cis retinal. Mutations can render the receptor active even in the absence of light. This constitutive activity can desensitize rod photoreceptor cells and lead to night blindness. A G90D mutation in rhodopsin causes the receptor to be constitutively active and leads to congenital stationary night blindness, which is generally thought to be devoid of retinal degeneration. The constitutively active species responsible for the night blindness phenotype is unclear. Moreover, the classification as a stationary disease devoid of retinal degeneration is also misleading. A transgenic mouse model for congenital stationary night blindness that expresses the G90D rhodopsin mutant was examined to better understand the origin of constitutive activity and the potential for retinal degeneration. Heterozygous mice for the G90D mutation did not exhibit retinal degeneration whereas homozygous mice exhibited progressive retinal degeneration. Only a modest reversal of retinal degeneration was observed when transducin signaling was eliminated genetically, indicating that some of the retinal degeneration occurred in a transducin-independent manner. Biochemical studies on purified rhodopsin from mice indicated that multiple species can potentially contribute to the constitutive activity causing night blindness.

Differential Aggregation Properties of Mutant Human and Bovine Rhodopsin.

Rhodopsin is the light receptor required for the function and health of photoreceptor cells. Mutations in rhodopsin can cause misfolding and aggregation of the receptor, which leads to retinal degeneration. Bovine rhodopsin is often used as a model to understand the effect of pathogenic mutations in rhodopsin due to the abundance of structural information on the bovine form of the receptor. It is unclear whether or not the bovine rhodopsin template is adequate in predicting the effect of these mutations occurring in human retinal disease or in predicting the efficacy of therapeutic strategies. To better understand the extent to which bovine rhodopsin can serve as a model, human and bovine P23H rhodopsin mutants expressed heterologously in cells were examined. The aggregation properties and cellular localization of the mutant receptors were determined by Förster resonance energy transfer and confocal microscopy. The potential therapeutic effects of the pharmacological compounds 9-cis retinal and metformin were also examined. Human and bovine P23H rhodopsin mutants exhibited different aggregation properties and responses to the pharmacological compounds tested. These observations would lead to different predictions on the severity of the phenotype and divergent predictions on the benefit of the therapeutic compounds tested. The bovine rhodopsin template does not appear to adequately model the effects of the P23H mutation in the human form of the receptor.

Lower PDL1, PDL2, and AXL Expression on Lung Myeloid Cells Suggests Inflammatory Bias in Smoking and Chronic Obstructive Pulmonary Disease.

Lung myeloid cells are important in pulmonary immune homeostasis and in the pathogenesis of chronic obstructive pulmonary disease (COPD). Multiparameter immunophenotypic characterization of these cells is challenging because of their autofluorescence and diversity. We evaluated the immunophenotypic landscape of airway myeloid cells in COPD using time of flight mass cytometry. Cells from BAL, which were obtained from never-smokers (n = 8) and smokers with (n = 20) and without (n = 4) spirometric COPD, were examined using a 44-parameter time of flight mass cytometry panel. Unsupervised cluster analysis was used to identify cellular subtypes that were confirmed by manual gating. We identified major populations of CD68+ and CD68- cells with 22 distinct phenotypic clusters, of which 18 were myeloid cells. We found a higher abundance of putative recruited myeloid cells (CD68+ classical monocytes) in BAL from patients with COPD. CD68+ classical monocyte population had distinct responses to smoking and COPD that were potentially related to their recruitment from the interstitium and vasculature. We demonstrate that BAL cells from smokers and subjects with COPD have lower AXL expression. Also, among subjects with COPD, we report significant differences in the abundance of PDL1high and PDL2high clusters and in the expression of PDL1 and PDL2 across several macrophage subtypes suggesting modulation of inflammatory responses. In addition, several phenotypic differences in BAL cells from subjects with history of COPD exacerbation were identified that could inform potential disease mechanisms. Overall, we report several changes to the immunophenotypic landscape that occur with smoking, COPD, and past exacerbations that are consistent with decreased regulation and increased activation of inflammatory pathways.

Effects of early pregnancy on uterine lymphocytes and endometrial expression of immune-regulatory molecules in dairy heifers.

Natural killer (NK) cells are essential for establishment of human and rodent pregnancies. The function of these and other cytotoxic T cells (CTL) is controlled by stimulatory and inhibitory signaling. A role for cytotoxic cells during early pregnancy in cattle has not been described, but regulation of their function at the fetal-maternal interface is thought to be critical for conceptus survival. The hypothesis that the relative abundance of CTL and expression of inhibitory signaling molecules is increased by the conceptus during early pregnancy was tested. The proportions of lymphoid lineage cells and expression of inhibitory signaling molecules in the endometrium during early pregnancy in dairy heifers were determined using flow cytometry, immunofluorescence, and real-time PCR on days 17 and 20 of pregnancy and day 17 of the estrous cycle. Results revealed an increased percentage of NKp46+ and CD8+ cells in the uterus of pregnant heifers. Furthermore, a large percentage of uterine immune cells coexpressed these proteins. Compared to cyclic heifers, CD45+ uterine cells from pregnant heifers exhibited greater degranulation. Endometrium from pregnant heifers had greater mRNA abundance for the inhibitory molecules, CD274 and lymphocyte activating gene 3 (LAG3), and greater cytotoxic T lymphocyte-associated protein 4 (CTLA4), molecules that can interact with receptors on antigen-presenting cells and induce lymphocyte tolerance. This study demonstrates a dynamic regulation of both cytotoxic immune cells and tolerogenic molecules during the peri-implantation period that may be required to support establishment of pregnancy and placentation.

Embryo Mortality Around the Period of Maintenance of the Corpus Luteum Causes Alterations to the Ovarian Function of Lactating Dairy Cows.

Objectives were to identify cows with embryo mortality (EM) around the period of corpus luteum maintenance by interferon tau (IFNT) and to characterize ovarian function in cows that underwent EM. Lactating Holstein cows received artificial insemination (AI) (Day = 0) with semen or extender only. From Day 14 to 42 transrectal ultrasonography was performed daily to monitor ovarian dynamics and uterine contents whereas blood was collected every 48 h to determine ISG15 and MX2 mRNA abundance in blood mononuclear cells (Day 14 to 22 only) and determination of hormone concentrations. Cows were classified in the following reproductive status groups: cyclic (inseminated with extender; n = 15), pregnant (embryo present on Day 42; n = 23), no embryo (n = 23), and EM (n = 14). EM was defined as the presence of an embryo based on interferon-stimulated genes (ISG) mRNA abundance and concentrations of pregnancy-specific protein B (PSPB) above specific cutoff points but no embryo visualized by ultrasonography. Within the EM group, early EM (up to Day 22) was when ISG fold changes were above specific cutoff points from Day 18 to 22 and PSPB below 0.7 ng/ml on and after Day 24, whereas late EM (after Day 22) was when PSPB was above 0.7 ng/ml on or after Day 24 regardless of ISG expression. This experiment provided evidence that the combination of ISG expression patterns and PSPB concentrations is a reasonable method to determine EM around the period of corpus luteum maintenance by IFNT because cows with evidence of EM had patterns of ISG expression more similar to pregnant than cyclic cows or cows with no embryo. Within the EM group, only cows with late EM had delayed luteal regression and longer interovulatory intervals. No major alterations in follicular function were observed after the onset of luteolysis. Our results suggest that embryo development needs to continue beyond 22 days after AI to effectively prevent luteolysis and extend the luteal phase.

Changes in Myeloid Lineage Cells in the Uterus and Peripheral Blood of Dairy Heifers During Early Pregnancy.

Establishment of pregnancy requires interaction between the developing conceptus and the uterine mucosal immune system. Myeloid lineage cells (macrophages and dendritic cells) are key mediators of pregnancy in rodents and humans but relatively little is known regarding their role and distribution during early pregnancy in ruminants. We tested the hypothesis that myeloid lineage cell number, distribution, and function are altered during early pregnancy in dairy heifers. Dairy heifers were inseminated using sperm from a single bull (Day 0), and uteri and blood were collected at slaughter on Days 17 and 20 of pregnancy to investigate the response of myeloid lineage cells to the presence of a conceptus. Responses were compared to noninseminated heifers on Day 17 of the estrous cycle. Peripheral blood and uterine-derived immune cells were isolated magnetically and examined using flow cytometry. Immunohistochemical analysis was used to evaluate the spatial distribution of myeloid lineage cells in the endometrium and quantitative polymerase chain reaction was conducted to quantify abundance of mRNA transcripts associated with myeloid lineage cell function. Transcripts for major histocompatibility complex (MHC) II, cluster of differentiation (CD) 80, CD86, CD163, and indoleamine 2,3-dioxygenase (IDO) 1 were greater in endometrium of pregnant compared to cyclic heifers. Immunofluorescence analysis revealed increased labeling for MHCII and SIRPA in pregnant compared to cyclic heifers. There were approximately 50% more CD14+CD11c+ cells in the peripheral circulation of pregnant compared to cyclic heifers. A greater number of myeloid lineage cells were observed during early pregnancy, and this increase was most pronounced in and around the shallow glands. Furthermore, expression of molecules associated with a tolerogenic or alternatively activated phenotype of these cells also increased in pregnant heifers. The results support the hypothesis that myeloid lineage cells with a tolerogenic phenotype are involved in establishment of pregnancy in dairy heifers.

Aggregation of rhodopsin mutants in mouse models of autosomal dominant retinitis pigmentosa.

Mutations in rhodopsin can cause it to misfold and lead to retinal degeneration. A distinguishing feature of these mutants in vitro is that they mislocalize and aggregate. It is unclear whether or not these features contribute to retinal degeneration observed in vivo. The effect of P23H and G188R misfolding mutations were examined in a heterologous expression system and knockin mouse models, including a mouse model generated here expressing the G188R rhodopsin mutant. In vitro characterizations demonstrate that both mutants aggregate, with the G188R mutant exhibiting a more severe aggregation profile compared to the P23H mutant. The potential for rhodopsin mutants to aggregate in vivo was assessed by PROTEOSTAT, a dye that labels aggregated proteins. Both mutants mislocalize in photoreceptor cells and PROTEOSTAT staining was detected surrounding the nuclei of photoreceptor cells. The G188R mutant promotes a more severe retinal degeneration phenotype and greater PROTEOSTAT staining compared to that promoted by the P23H mutant. Here, we show that the level of PROTEOSTAT positive cells mirrors the progression and level of photoreceptor cell death, which suggests a potential role for rhodopsin aggregation in retinal degeneration.

NF-κB inhibitor alpha controls SARS-CoV-2 infection in ACE2-overexpressing human airway organoids.

As SARS-CoV-2 continues to spread worldwide, tractable primary airway cell models that recapitulate the cell-intrinsic response to arising viral variants are needed. Here we describe an adult stem cell-derived human airway organoid model overexpressing the ACE2 receptor (ACE2-OE) that supports robust viral replication while maintaining 3D architecture and cellular diversity of the airway epithelium. ACE2-OE organoids were infected with SARS-CoV-2 variants and subjected to single-cell RNA-sequencing. Interferon-lambda was upregulated in cells with low-level infection while the NF-kB inhibitor alpha gene (encoding IkBa) was consistently upregulated in infected cells, and its expression positively correlated with infection levels. Confocal microscopy showed more IkBa expression in infected than bystander cells, but found concurrent nuclear translocation of NF-kB that IkBa usually prevents. Overexpressing a nondegradable IkBa mutant reduced NF-kB translocation and increased viral infection. These data demonstrate the functionality of ACE2-OE organoids in SARS-CoV-2 research and underscore that the strength of the NF-kB feedback loop in infected cells controls viral replication.

NF-κB inhibitor alpha has a cross-variant role during SARS-CoV-2 infection in ACE2-overexpressing human airway organoids.

As SARS-CoV-2 continues to spread worldwide, tractable primary airway cell models that accurately recapitulate the cell-intrinsic response to arising viral variants are needed. Here we describe an adult stem cell-derived human airway organoid model overexpressing the ACE2 receptor that supports robust viral replication while maintaining 3D architecture and cellular diversity of the airway epithelium. ACE2-OE organoids were infected with SARS-CoV-2 variants and subjected to single-cell RNA-sequencing. NF-κB inhibitor alpha was consistently upregulated in infected epithelial cells, and its mRNA expression positively correlated with infection levels. Confocal microscopy showed more IκBα expression in infected than bystander cells, but found concurrent nuclear translocation of NF-κB that IκBα usually prevents. Overexpressing a nondegradable IκBα mutant reduced NF-κB translocation and increased viral infection. These data demonstrate the functionality of ACE2-OE organoids in SARS-CoV-2 research and identify an incomplete NF-κB feedback loop as a rheostat of viral infection that may promote inflammation and severe disease.

The Transcriptomic Evolution of Mammalian Pregnancy: Gene Expression Innovations in Endometrial Stromal Fibroblasts.

The endometrial stromal fibroblast (ESF) is a cell type present in the uterine lining of therian mammals. In the stem lineage of eutherian mammals, ESF acquired the ability to differentiate into decidual cells in order to allow embryo implantation. We call the latter cell type "neo-ESF" in contrast to "paleo-ESF" which is homologous to eutherian ESF but is not able to decidualize. In this study, we compare the transcriptomes of ESF from six therian species: Opossum (Monodelphis domestica; paleo-ESF), mink, rat, rabbit, human (all neo-ESF), and cow (secondarily nondecidualizing neo-ESF). We find evidence for strong stabilizing selection on transcriptome composition suggesting that the expression of approximately 5,600 genes is maintained by natural selection. The evolution of neo-ESF from paleo-ESF involved the following gene expression changes: Loss of expression of genes related to inflammation and immune response, lower expression of genes opposing tissue invasion, increased markers for proliferation as well as the recruitment of FOXM1, a key gene transiently expressed during decidualization. Signaling pathways also evolve rapidly and continue to evolve within eutherian lineages. In the bovine lineage, where invasiveness and decidualization were secondarily lost, we see a re-expression of genes found in opossum, most prominently WISP2, and a loss of gene expression related to angiogenesis. The data from this and previous studies support a scenario, where the proinflammatory paleo-ESF was reprogrammed to express anti-inflammatory genes in response to the inflammatory stimulus coming from the implanting conceptus and thus paving the way for extended, trans-cyclic gestation.