Red Pulp Of Spleen Research Articles

Optimization of polyethylene glycol shielding and mannose density on the lipid nanoparticles for efficient delivery to macrophages and spleens

This study compared the effects of polyethylene glycol (PEG) shielding and mannose-conjugated ligands density on lipid nanoparticles (LNPs) for intracellular uptake to macrophages in vitro and accumulation in spleens in vivo. Fabricated phosphatidyl serine-incorporated LNPs (sLNPs) was physically decorated with mannose-conjugated DSPE-PEG (DPM) at different DPM/LNP molar ratios achieving the DPM density from 0 to 0.6 PEGs/nm2. We demonstrated that low PEG shielding sLNPs with mannose ligands (sLNP-DPMs) displayed superior uptake to macrophages (RAW 264.7 cells) compared with high PEG shielding sLNP-DPMs in vitro. However, high PEG shielding sLNP-DPMs showed significant spleen accumulation compared with low PEG shielding sLNP-DPMs in vivo after intravenous injection. In particular, high PEG shielding sLNPs coated with DSPE-methoxyPEG (DP) and DPM mixture at DP/DPM molar ratios of 5/5 exhibited greater accumulation in red pulp of spleens than naked sLNPs by 2.7-folds in vivo. These results suggested that the optimal PEG shielding and mannose densities per a particle might be different between in vitro cellular uptake to macrophages and in vivo spleen accumulation after systemic administration. Taken together, precision-tailored LNP-surface modifications achieved through optimization of PEG shielding and mannose density can greatly enhance accumulation of LNPs in red pulp of spleens, which could be applied for the delivery of nucleic acid-based drugs and vaccines to spleens in vivo.

Glomerular Hypertrophy and Splenic Red Pulp Degeneration Concurrent with Oxidative Stress in 3xTg-AD Mice Model for Alzheimer's Disease and Its Exacerbation with Sex and Social Isolation.

The continuously expanding field of Alzheimer's disease (AD) research is now beginning to defocus the brain to take a more systemic approach to the disease, as alterations in the peripheral organs could be related to disease progression. One emerging hypothesis is organ involvement in the process of Aβ clearance. In the present work, we aimed to examine the status and involvement of the kidney as a key organ for waste elimination and the spleen, which is in charge of filtering the blood and producing lymphocytes, and their influence on AD. The results showed morphological and structural changes due to acute amyloidosis in the kidney (glomeruli area) and spleen (red pulp area and red/white pulp ratio) together with reduced antioxidant defense activity (GPx) in 16-month-old male and female 3xTg-AD mice when compared to their age- and sex-matched non-transgenic (NTg) counterparts. All these alterations correlated with the anxious-like behavioral phenotype of this mouse model. In addition, forced isolation, a cause of psychological stress, had a negative effect by intensifying genotype differences and causing differences to appear in NTg animals. This study further supports the relevance of a more integrative view of the complex interplay between systems in aging, especially at advanced stages of Alzheimer's disease.

MALDI Imaging Mass Spectrometry Visualizes the Distribution of Antidepressant Duloxetine and Its Major Metabolites in Mouse Brain, Liver, Kidney, and Spleen Tissues.

Imaging mass spectrometry (IMS) is a powerful tool for mapping the spatial distribution of unlabeled drugs and metabolites that may find application in assessing drug delivery, explaining drug efficacy, and identifying potential toxicity. This study focuses on determining the spatial distribution of the antidepressant duloxetine, which is widely prescribed despite common adverse effects (liver injury, constant headaches) whose mechanisms are not fully understood. We used high-resolution IMS with matrix-assisted laser desorption/ionization to examine the distribution of duloxetine and its major metabolites in four mouse organs where it may contribute to efficacy or toxicity: brain, liver, kidney, and spleen. In none of these tissues is duloxetine or its metabolites homogeneously distributed, which has implications for both efficacy and toxicity. We found duloxetine to be similarly distributed in spleen red pulp and white pulp but differentially distributed in different anatomic regions of the liver, kidney, and brain, with dose-dependent patterns. Comparison with hematoxylin and eosin staining of tissue sections reveals that the ion images of endogenous lipids help delineate anatomic regions in the brain and kidney, while heme ion images assist in differentiating regions within the spleen. These endogenous metabolites may serve as a valuable resource for examining the spatial distribution of other drugs in tissues when staining images are not available. These findings may facilitate future mechanistic studies of the therapeutic and adverse effects of duloxetine. In the current work, we did not perform absolute quantification of duloxetine, which will be reported in due course. SIGNIFICANCE STATEMENT: The study utilized imaging mass spectrometry to examine the spatial distribution of duloxetine and its primary metabolites in mouse brain, liver, kidney, and spleen. These results may pave the way for future investigations into the mechanisms behind duloxetine's therapeutic and adverse effects. Furthermore, the mass spectrometry images of specific endogenous metabolites such as heme could be valuable in analyzing the spatial distribution of other drugs within tissues in scenarios where histological staining images are unavailable.

Morphological examination of rat organs with T-2 toxicosis against the background of the use of the complex preventive agent "Zeapitox"

It is impossible to find out the true state of the tissue organization without conducting morphological studies. In this regard, histological analysis very objectively demonstrates the structure of cellular compositions, which is especially in demand when making a reliable diagnosis and monitoring therapeutic and preventive effectiveness. T-2 toxin, as a biogenic poison, poses a potentially permanent threat to human and animal health. It is also important that modern highly productive breeds of farm animals and bird crosses have an increased sensitivity to mycotoxins. In order to prevent mycotoxicosis, a selection of diets containing sorbents and adaptogens is promising in terms of economic and therapeutic effectiveness. The purpose of the experiment was to evaluate the preventive effectiveness of «Zeapitox» from a morphological point of view when rats were poisoned with T-2 toxin at the tissue level of a number of organs. The experiment involved 30 nonlinear rats, males weighing in the range of 190-210 grams. The first group served as biological control; individuals of the second group were given T-2 toxin at a dose of 1/10 of LD50; animals of the third group, in addition to T-2 toxin at a dose of 1/10 of LD50, were additionally fed at a dose of 0,5 % of the diet with the feed «Zeapitox». Under the influence of T-2 toxin on the histostructure of rat organ tissues, the following changes were registered: hyperemia, swelling, increased blood filling of blood vessels, as well as destructive processes of the central lobules of hepatocytes, intestinal epithelium and blood cells. The protective effect of the complex preventiveagent «Zeapitox» is manifested by normal blood filling in the spleen (red and white pulp), native beam structure of lobules and good visualization without pathological phenomena of hepatocytes, absence of edematous processes in the mucous membranes of intestinal.

Correction of morphofunctional changes in mice thymus and spleen by Rhaponticum uniflorum (L.) DC. extract at cyclophosphamide immunosupression

The aim of the study is to evaluate the effect of R. uniflorum dry extract on morphofunctional state of mice thymus and spleen at cyclophosphamide immunosuppression.Materials and methods. The experiments were carried out on F1 (CBAxC57Bl/6) mice 18–20 g of weight. Immune deficiency was simulated by a single intraperitoneal introduction of cyclophosphamide in the dose 250 mg/kg. R. uniflorum dry extract in the dose 100 mg/kg was administered per os for 14 days. The thymus and spleen were fixed for histological studies on day 16. The thymus total area, the cortex and medulla area, the connective tissue capsule thickness, the spleen white and red pulp area were used to measure on micropreparations stained with hematoxylineosin and azure-eosin. The cell density and the cellular composition were calculated in the thymus cortical substance.Results. The R. uniflorum extract increased the thymus total area and the corticomedullary index by 37 and 26 % (p ≤ 0.05), respectively. The cells density increased in the cortical substance, it decreased in the medulla, as a result “layer inversion” is not observed in any animal of the experimental group. The number of proliferating lymphocytes, blasts and large lymphocytes increased; the number of destructive cells and macrophages decreased in the cortical substance. The spleen white pulp relative volume was 39 % higher than in the control.Conclusions. The R. uniflorum dry extract had an immunocorrective effect on the thymus and spleen morphofunctional state at cyclophosphamid immunosuppression.

Thymic macrophages consist of two populations with distinct localization and origin.

Tissue-resident macrophages are essential to protect from pathogen invasion and maintain organ homeostasis. The ability of thymic macrophages to engulf apoptotic thymocytes is well appreciated, but little is known about their ontogeny, maintenance, and diversity. Here, we characterized the surface phenotype and transcriptional profile of these cells and defined their expression signature. Thymic macrophages were most closely related to spleen red pulp macrophages and Kupffer cells and shared the expression of the transcription factor SpiC with these cells. Single-cell RNA sequencing showed that the macrophages in the adult thymus are composed of two populations distinguished by the expression of Timd4 and Cx3cr1. Remarkably, Timd4+ cells were located in the cortex, while Cx3cr1+ macrophages were restricted to the medulla and the cortico-medullary junction. Using shield chimeras, transplantation of embryonic thymuses, and genetic fate mapping, we found that the two populations have distinct origins. Timd4+ thymic macrophages are of embryonic origin, while Cx3cr1+ macrophages are derived from adult hematopoietic stem cells. Aging has a profound effect on the macrophages in the thymus. Timd4+ cells underwent gradual attrition, while Cx3cr1+ cells slowly accumulated with age and, in older mice, were the dominant macrophage population in the thymus. Altogether, our work defines the phenotype, origin, and diversity of thymic macrophages.

Cadmium-induced splenic lymphocytes anoikis is not mitigated by activating Nrf2-mediated antioxidative defense response

Cadmium (Cd) is a widely used heavy metal which is reported to exert extensive harm to the environment and human health. Owing to Cd being an element it is continuously enriched in the environment. The mechanism of splenic toxicity by Cd, however, is not yet clear. In order to explore the toxic mechanism of Cd exposure to the spleen, we added 0, 35, 70 and 140 mg/kg of Cd to the diet of chicken and fed them this diet for 90 days. Analysis of histopathological sections showed that Cd exposure damaged the spleen structure, the spleen red pulp, the white pulp boundary disappeared and the number of lymphocytes decreased significantly, suggesting that Cd exposure leads to organ injury to the spleen. Particularly, Cd-induced anoikis - a special form of programmed cell death caused by the loss of contact between cells and extracellular matrix and other cells - is associated with integrin-related cell detachment and activation of apoptotic signaling pathways. Moreover, Cd exposure leads to an increase in free radicals content and affects the activity of antioxidant enzymes resulting in oxidative stress. Simultaneously, Cd activated the body's antioxidant defense system mediated by the Nuclear factor related factor 2 (Nrf2) signaling pathway. Based on our results Cd-induced splenic lymphocytes anoikis is not mitigated by Nrf2-mediated antioxidative defense response.

Time-dependent biodistribution profiles and reaction of polyethylene glycol-coated iron oxide nanoclusters in the spleen after intravenous injection in the mice

Polyethylene glycol (PEG) is widely used polymer in the field of pharmaceutics, particularly in which related to drug delivery systems (DDS). Surface coating of the nanoparticles (NPs) with PEG (i.e. pegylation) adds novel characteristics that make their use in vivo more effective with lower cytotoxicity. The biodistribution profiles, reaction, and fate of PEG-coated NPs in vivo still unclear and need more detailed studies. Here in this study, we prepared PEG-coated iron oxide nanoclusters (PEG-coated IONCs) to investigate their biodistribution profiles and reactions in spleen after intravenous injection time-dependently. Using Prussian blue staining method as specific histochemical reaction for iron detection in the tissues, the PEG-coated IONCs were observed in a higher ratio in spleen red pulp after 1 day of injection but decreased time-dependently after 10 days and 20 days. Interestingly, PEG-coated IONCs moved from red pulp into the white pulp specially after 20 days of injection. After long time exposure (20 days), higher amount of PEG-coated IONCs was observed in the center of spleen white pulp follicle. Using histological staining, the reaction of PEG-coated IONCs with splenocytes or immune cells induced cellular abnormalities such as, nucleic acid damages, induction of megakaryocytes number, and sever vacuolation in the white pulp area specially after 20 days of injection. Histochemically, the localization of PEG-coated IONCs in the splenic parenchyma induced the level of the collagen fibers particularly after 1 day and 10 days of injection. Interestingly, cellular abnormalities in the splenic red pulp as well as collagen levels decreased after 20 days of injection due to the clearance of PEG-coated IONCs from this area. This data indicated that cytotoxicity produced by the reaction of PEG-coated IONCs in the spleen are reversible specially after 20 days of in the intravenous injection. Understanding the detailed mechanism of the fate and reaction of the coated nanomaterials after intravenous injection is important to design effective and safe DDS based NPs.

A higher dose of PEGylated gold nanoparticles reduces the accelerated blood clearance phenomenon effect and induces spleen B lymphocytes in albino mice.

Polyethylene glycol (PEG) is a multifunctionalpolymer that has many uses in medical and biological applications. Recently, PEG has been mainly used in developing nanomaterial-based drug delivery systems (DDS). PEG is characterized by its high solubility, biological inertness, and ability to escape from immune cells (stealthiness) after systemic injection. The most challenging problem for PEGylated nanomaterials is their rapid elimination from the bloodstream after repeated doses of systemic injection, called accelerated blood clearance (ABC). Therefore, in this study, the effect of PEGylated nanomaterial dose concentration on ABC induction will be investigated using quantitative, histological, and immunohistochemical analyses. A higher dose concentration (2mg/kg) of PEGylated gold nanoparticles (PEG-coated AuNPs) reduced the ABC phenomenon when intravenously injected into mice preinjected with the same dose. In contrast, a lower dose concentration (< 1mg/kg) significantly induced the ABC phenomenon by the rapid elimination of the second dose of PEG-coated AuNPs from the bloodstream. To explain the relationship between the dose concentration (from PEG and AuNPs) and the induction of ABC, the biodistribution of PEG-coated AuNPs in liver and spleen [reticuloendothelial systems (RES)-rich organs] was investigated. The injected dose of PEG-coated AuNPs accumulated mainly in the hepatic Kupffer cells and hepatocytes. Similarly, spleen red pulp received a higher amount of the injected dose of PEG-coated AuNPs. However, the biodistriution profiles of PEG-coated AuNPs after the first and second dose for different dose concentrations varied in RES-rich organs. Additionally, the number of B lymphocytes, which have an important role in producing anti-PEG immunoglobulin (Ig)M, was affected by the repeated dose of PEG-coated AuNPs in the spleen. Therefore, for effective nanomaterial-based DDS development, dose optimization of PEG molecules that express PEGylated nanomaterials is important to reduce the ABC phenomenon effect. The ideal concentration of PEG molecules used to coat nanomaterials and the role of RES-rich organs must be determined to control the ABC phenomenon effect of PEGylated nanomaterials.

Genetic Influence on Frequencies of Myeloid-Derived Cell Subpopulations in Mouse.

Differences in frequencies of blood cell subpopulations were reported to influence the course of infections, atopic and autoimmune diseases, and cancer. We have discovered a unique mouse strain B10.O20 containing extremely high frequency of myeloid-derived cells (MDC) in spleen. B10.O20 carries 3.6% of genes of the strain O20 on the C57BL/10 genetic background. It contains much higher frequency of CD11b+Gr1+ cells in spleen than both its parents. B10.O20 carries O20-derived segments on chromosomes 1, 15, 17, and 18. Their linkage with frequencies of blood cell subpopulations in spleen was tested in F2 hybrids between B10.O20 and C57BL/10. We found 3 novel loci controlling MDC frequencies: Mydc1, 2, and 3 on chromosomes 1, 15, and 17, respectively, and a locus controlling relative spleen weight (Rsw1) that co-localizes with Mydc3 and also influences proportion of white and red pulp in spleen. Mydc1 controls numbers of CD11b+Gr1+ cells. Interaction of Mydc2 and Mydc3 regulates frequency of CD11b+Gr1+ cells and neutrophils (Gr1+Siglec-F- cells from CD11b+ cells). Interestingly, Mydc3/Rsw1 is orthologous with human segment 6q21 that was shown previously to determine counts of white blood cells. Bioinformatics analysis of genomic sequence of the chromosomal segments bearing these loci revealed polymorphisms between O20 and C57BL/10 that change RNA stability and genes’ functions, and we examined expression of relevant genes. This identified potential candidate genes Smap1, Vps52, Tnxb, and Rab44. Definition of genetic control of MDC can help to personalize therapy of diseases influenced by these cells.

Anti-Leishmania infantum Antibody-Producing Plasma Cells in the Spleen in Canine Visceral Leishmaniasis.

The spleen is involved in visceral leishmaniasis immunopathogenesis, and presents alterations in white-pulp microenvironments that are associated with an increased susceptibility to coinfections and patient death. Plasmacytosis in splenic red pulp (RP) is one observed alteration, but the specificity of antibody-secreting cells and the distribution of them has not yet been evaluated. We biotinylated soluble L. infantum membrane antigens (bSLMA) used as probes in modified immunohistochemistry, and detected the presence of anti-L. infantum antibody-secreting cells. Were used spleens from eight dogs from the endemic area for canine visceral leishmaniasis (CanL), and three healthier controls. The spleen sections were cryopreserved, and we performed modified immunohistochemistry. The ratio of plasma cells which were reactive to bSLMA (Anti-Leish-PC) in the spleen RP and periarteriolar lymphatic sheath (PALS) were calculated. Dogs with CanL present hyperglobulinemia and more plasma cells in their RP than the controls. Furthermore, dogs with CanL presented a lower proportion of Anti-Leish-PC in their RP than in PALS. Likewise, dysproteinemia was related to RP and PALS plasmacytosis, and a more severe clinical profile.

Clinical signs, lethal dose and histopathological lesions in grass carp, Ctenopharyngodon idella experimentally infected with Edwardsiella tarda

An experiment was conducted to study the lethal dose (LD50-96h) and histopathological changes occurring in several organs of grass carp challenged with different concentrations of Edwardsiella tarda. The healthy grass carps were challenged with the bacterial suspension of 106,107, 108, 109 and 1010 CFU ml−1. The study demonstrated that the lethal dose (LD50-96h) of E. tarda for grass carp is 1.3 × 109 CFU ml−1. The infected fish showed abnormal swimming behavior, slower movements, skin necrosis, hemorrhages, and open lesion on the fontanelle of the frontal bone of the skull during the initial phase of infection. About 60% of the fish which received the bacterial suspension of 1010 CFU ml−1 died within 24 h of infection. The histopathological examination of the infected tissue section demonstrated the severe damages in the internal organs. In gills, oedema, secondary lamellae fusion, and hyperplasia of basal epithelial lining between secondary lamellae were reported. The microscopic observation showed the disruption of submucosa to the mucosa, which finally led to degenerative changes in the intestine, necrosis of hepatocytes and infiltration of red blood cells in the liver. The tubular disintegration in kidney and loss of capsular boundary of red pulp in spleen were also reported. In conclusion, the result indicates that the infection caused by E. tarda can cause severe damages and alterations in grass carp tissues and potential mass mortality. Moreover, The bacteria isolated from the mobribund fish was characterized by biochemical tests and expression of five critical virulence genes like citC, fimA, gadB, mukF and gyrB were detected from the microorganism. The study aims to provide a research foundation for further studies on the susceptibility and pathological changes of grass carp induced by E. tarda infection.

Abstract 529: Characterization of a humanized TLR8 mouse model and its application in immuno-oncology drug development

Abstract Background: TLR8 belongs to the Toll-like receptor family, serving as endosomal sensors of uridine rich single strand viral or endogenous RNA and several types of small molecule agonist. In humans, TLR8 is primarily expressed on monocytes, macrophages and myeloid dendritic cells (DCs), while in murine it was originally reported that TLR8 has no function due to the lack of five amino acids that recognize the endogenous RNA ligand. Increasing evidence suggests that mouse TLR8 may function differently from its human counterpart, including ligand binding and signaling through pathways other than NK-κB. Therefore, a humanized TLR8 mouse model is pivotal for mechanistically understanding TLR8 innate immune regulation and pharmacologically targeting TLR8 in various malignancies and inflammatory diseases. Methods: Exon 3 of mouse Tlr8 was replaced with the corresponding human region to generate humanized TLR8 knock-in mice. hTLR8+/- mice (n=5) and age matched naïve C57BL/6 mice were maintained until 27 weeks of age. Liver, spleen, kidney and pancreas were collected for pathological characterization. Serum cytokines levels were analyzed by MSD, and hTLR8 expression pattern and immune cells were analyzed in the spleen by flow cytometry. Comparisons of anti-PD-1 response in TLR8 knock-in mice and naïve C57BL/6 mice bearing various syngeneic tumors were also conducted. Results: We systemically characterized the phenotypic changes between age-matched 27-week-old C57BL/6 and hTLR8 mice. A high percentage of hTLR8 expression was detected on CD11b+ F4/80+ macrophages, CD11b+Gr-1+ myeloid cells and CD11c+ DCs. Hepatomegaly, splenomegaly, and nephromegaly were observed in aged hTLR8 mice. Histology analysis revealed a prominent portal inflammation with fibrosis and bile duct hyperplasia in liver; a decreased cellularity in periarteriolar lymphoid sheath and increase of histiocytes in the red pulp of spleen; and inflammatory immune cell infiltration in the kidneys. Analysis of serum cytokines showed a significant increase of KC/GRO (CXLCL1), TNF-α and IL-10 in the peripheral blood of the aged hTLR8 mice. PBMC from hTLR8 mice responded to a specific RNA-ligand based human TLR8 agonist (ORN-8L) in vitro. We are now profiling different syngeneic and genetically engineered murine tumor models to fully characterize the potential adjuvant-like function of hTLR8 in different tumor models. Conclusion: Expression of humanized TLR8 leads to auto-activation of TLR8 signaling in mice. This pivotal model system enables us to study the function of human TLR8 and pharmacology of its agonists and antagonists in vivo. These knock-in mice may also serve as an interesting host strain for syngeneic tumor studies, potentiating immune responses to therapies. Citation Format: Hongjuan Zhang, Ruilin Sun, Annie X. An, Henry Q.x Li, Davy Xuesong Ouyang. Characterization of a humanized TLR8 mouse model and its application in immuno-oncology drug development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 529.

Who am I? (re-)Defining fibroblast identity and immunological function in the age of bioinformatics.

Who am I? (re-)Defining fibroblast identity and immunological function in the age of bioinformatics.

Combining microfluidics with machine learning algorithms for RBC classification in rare hereditary hemolytic anemia

Combining microfluidics technology with machine learning represents an innovative approach to conduct massive quantitative cell behavior study and implement smart decision-making systems in support of clinical diagnostics. The spleen plays a key-role in rare hereditary hemolytic anemia (RHHA), being the organ responsible for the premature removal of defective red blood cells (RBCs). The goal is to adapt the physiological spleen filtering strategy for in vitro study and monitoring of blood diseases through RBCs shape analysis. Then, a microfluidic device mimicking the slits of the spleen red pulp area and video data analysis are combined for the characterization of RBCs in RHHA. This microfluidic unit is designed to evaluate RBC deformability by maintaining them fixed in planar orientation, allowing the visual inspection of RBC’s capacity to restore their original shape after crossing microconstrictions. Then, two cooperative learning approaches are used for the analysis: the majority voting scheme, in which the most voted label for all the cell images is the class assigned to the entire video; and the maximum sum of scores to decide the maximally scored class to assign. The proposed platform shows the capability to discriminate healthy controls and patients with an average efficiency of 91%, but also to distinguish between RHHA subtypes, with an efficiency of 82%.

EpoR-tdTomato-Cre mice enable identification of EpoR expression in subsets of tissue macrophages and hematopoietic cells

AbstractThe erythropoietin receptor (EpoR) has traditionally been thought of as an erythroid-specific gene. Notably, accumulating evidence suggests that EpoR is expressed well beyond erythroid cells. However, the expression of EpoR in non-erythroid cells has been controversial. In this study, we generated EpoR-tdTomato-Cre mice and used them to examine the expression of EpoR in tissue macrophages and hematopoietic cells. We show that in marked contrast to the previously available EpoR-eGFPcre mice, in which a very weak eGFP signal was detected in erythroid cells, tdTomato was readily detectable in both fetal liver (FL) and bone marrow (BM) erythroid cells at all developmental stages and exhibited dynamic changes during erythropoiesis. Consistent with our recent finding that erythroblastic island (EBI) macrophages are characterized by the expression of EpoR, tdTomato was readily detected in both FL and BM EBI macrophages. Moreover, tdTomato was also detected in subsets of hematopoietic stem cells, progenitors, megakaryocytes, and B cells in BM as well as in spleen red pulp macrophages and liver Kupffer cells. The expression of EpoR was further shown by the EpoR-tdTomato-Cre–mediated excision of the floxed STOP sequence. Importantly, EPO injection selectively promoted proliferation of the EpoR-expressing cells and induced erythroid lineage bias during hematopoiesis. Our findings imply broad roles for EPO/EpoR in hematopoiesis that warrant further investigation. The EpoR-tdTomato-Cre mouse line provides a powerful tool to facilitate future studies on EpoR expression and regulation in various non-hematopoietic cells and to conditionally manipulate gene expression in EpoR-expressing cells for functional studies.

Heterogenicity of Tissue Distribution Recognized by A panel of Monoclonal Antibodies

Abstract ACE2 (Angiotensin Converting Enzyme 2) is the main viral entry point for SARS-CoV and SARS-CoV-2. Human ACE2 is widely expressed on different organs but if virus attached all the organs that highly expressed ACE2 is not fully studied. Most recent finding showed that a novel short isoform of ACE2 expressed in the airway epithelium is substantially upregulated in response to interferon stimulation, but not SARS-CoV-2 infection due to this short isoform lacking SARS-CoV-2 spike high-affinity binding sites. This arouses our interest in whether ACE2 has more isoforms expressed in different tissues that directly affect different organs response to virus infection. To support understanding ACE2 expression in response to viral infection, we generated human ACE2 monoclonal antibody by immunizing rat with recombinant human ACE2 protein covering extracellular domain (Gln18-Ser740). We found that there is discrepancy in different clones on different tissues. After three rounds subcloning, four clones have been selected based on chromosomally stable IgG producing hybridoma cells. Our data showed that all four clones showed equivalent signals on ACE2 transfectants but staining signals varied on tubules of kidney and seminiferous tubules and leyding cells on testis, two clones showed medium level staining signals on intestine and colon, but only one clone showed positive staining signals on spleen vascular and red pulp sinus endothelium. In addition, different neutralization potency and Western blot detection performance were observed among the collections of clones. Our data indicated that different clones might recognize different epitopes of ACE2 and indirectly suggested that ACE2 might have different isoforms in different tissues.

CD49d marks Th1 and Tfh-like antigen-specific CD4+ T cells during Plasmodium chabaudi infection.

Upon activation, specific CD4+ T cells up-regulate the expression of CD11a and CD49d, surrogate markers of pathogen-specific CD4+ T cells. However, using T-cell receptor transgenic mice specific for a Plasmodium antigen, termed PbT-II, we found that activated CD4+ T cells develop not only to CD11ahiCD49dhi cells, but also to CD11ahiCD49dlo cells during acute Plasmodium infection. CD49dhi PbT-II cells, localized in the red pulp of spleens, expressed transcription factor T-bet and produced IFN-γ, indicating that they were type 1 helper T (Th1)-type cells. In contrast, CD49dlo PbT-II cells resided in the white pulp/marginal zones and were a heterogeneous population, with approximately half of them expressing CXCR5 and a third expressing Bcl-6, a master regulator of follicular helper T (Tfh) cells. In adoptive transfer experiments, both CD49dhi and CD49dlo PbT-II cells differentiated into CD49dhi Th1-type cells after stimulation with antigen-pulsed dendritic cells, while CD49dhi and CD49dlo phenotypes were generally maintained in mice infected with Plasmodium chabaudi. These results suggest that CD49d is expressed on Th1-type Plasmodium-specific CD4+ T cells, which are localized in the red pulp of the spleen, and can be used as a marker of antigen-specific Th1 CD4+ T cells, rather than that of all pathogen-specific CD4+ T cells.

DCIR3 and DCIR4 are widely expressed among tissue-resident macrophages with the exception of microglia and alveolar macrophages

Dendritic cell inhibitory receptor 3 (DCIR3, Clec4a3) and dendritic cell inhibitory receptor 4 (DCIR4, Clec4a1) are C-type lectin receptors that belong to mouse dendritic cell immunoreceptor (DCIR) family. We recently showed that DCIR3 and DCIR4 are co-expressed on inflammatory and patrolling monocytes. In this study, we investigated the expression of DCIR3 and DCIR4 on tissue-resident macrophages. We found that spleen red pulp macrophages, liver Kupffer cells, large and small peritoneal macrophages and small intestinal macrophages expressed both DCIR3 and DCIR4. By contrast, lung alveolar macrophages expressed DCIR3 but not DCIR4 and brain microglia expressed neither DCIR3 nor DCIR4. Considerable part of tissue-resident macrophages are derived from embryonic precursors. We, therefore, examined the expression of DCIR3 and DCIR4 on the embryonic precursors. Yolk-sac macrophages from embryonic day (E) 8.5 embryos expressed both DCIR3 and DCIR4, while DCIR3 and DCIR4 were expressed on subpopulations of fetal liver monocytes from E14.5 embryos. Our results, together with previous data, indicate that the expression of DCIR3 and DCIR4 is widely shared by mononuclear phagocytes, including monocytes and macrophages, and that the expression of DCIR3 and DCIR4 on the embryonic precursors are not always retained by their progenies, suggesting that expression of DCIR3 and DCIR4 on tissue-resident macrophages might be regulated by environment of the tissues where the embryonic precursors differentiate into macrophages.

A Quantitative Approach to SIV Functional Latency in Brain Macrophages.

Lentiviruses are retroviruses that primarily infect myeloid cells, leading to acute inflammatory infections in many tissues particularly, lung, joints and the central nervous system (CNS). Acute infection by lentiviruses is followed by persistent/latent infections that are not cleared by the host immune system. HIV and SIV are lentiviruses that also infect CD4+ lymphocytes as well as myeloid cells in blood and multiple tissues. HIV infection of myeloid cells in brain, lung and heart cause tissue specific diseases as well as infect cells in gut, lymph nodes and spleen. AIDS dementia and other tissue specific disease are observed when infected individuals are immunosuppressed and the number of circulating CD4+ T cells declines to low levels. Antiretroviral therapy (ART) controls viral spread and dramatically changes the course of immunodeficiency and AIDS dementia. However, ART does not eliminate virus-infected cells. Brain macrophages contain HIV DNA and may represent a latent reservoir that persists. HIV latency in CD4+ lymphocytes is the main focus of current research and concern in efforts to eradicate HIV. However, a number of studies have demonstrated that myeloid cells in blood and tissues of ART suppressed individuals harbor HIV DNA. The resident macrophages in tissues such as brain (microglia), spleen (red pulp macrophages) and alveolar macrophages in lung are derived from the yolk sac and can self renew. The question of the latent myeloid reservoir in HIV has not been rigorously examined and its potential as a barrier to eradication been considered. Using a well characterized SIV ART suppressed, non-human primate (NHP) model, our laboratory developed the first quantitative viral outgrowth assay (QVOA) designed to evaluate latently infected CD4+ lymphocytes and more recently developed a similar protocol for the assessment of latently infected myeloid cells in blood and brain. Using an SIV ART model, it was demonstrated that myeloid cells in blood and brain harbor latent SIV that can be reactivated and produce infectious virus in vitro. These studies demonstrate for the first time that myeloid cells have the potential to be a latent reservoir of HIV that produces infectious virus that can be reactivated in the absence of ART and during HIV eradication strategies. Graphical Abstract.