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Extramedullary erythropoiesis is not expected in healthy adult mice, but erythropoietic gene expression was elevated in lineage-depleted spleen cells from Cd47−/− mice. Expression of several genes associated with early stages of erythropoiesis was elevated in mice lacking CD47 or its signaling ligand thrombospondin-1, consistent with previous evidence that this signaling pathway inhibits expression of multipotent stem cell transcription factors in spleen. In contrast, cells expressing markers of committed erythroid progenitors were more abundant in Cd47−/− spleens but significantly depleted in Thbs1−/− spleens. Single-cell transcriptome and flow cytometry analyses indicated that loss of CD47 is associated with accumulation and increased proliferation in spleen of Ter119−CD34+ progenitors and Ter119+CD34− committed erythroid progenitors with elevated mRNA expression of Kit, Ermap, and Tfrc. Induction of committed erythroid precursors is consistent with the known function of CD47 to limit the phagocytic removal of aged erythrocytes. Conversely, loss of thrombospondin-1 delays the turnover of aged red blood cells, which may account for the suppression of committed erythroid precursors in Thbs1−/− spleens relative to basal levels in wild-type mice. In addition to defining a role for CD47 to limit extramedullary erythropoiesis, these studies reveal a thrombospondin-1-dependent basal level of extramedullary erythropoiesis in adult mouse spleen.

The spleen is the largest lymphoid organ of the body, which participates in the regulation of metabolic balance. High altitude hypoxia environment can affect lipid metabolism in spleen tissue, but the key mechanism of lipid metabolism is still unclear. We aimed to use lipidomic analysis to study the effect of high altitude hypoxia on lipid metabolism in mouse spleen tissue. C57BL/6 mice were placed at an altitude of 4 200 m and 400 m, respectively, and after 30 days the spleen tissues were harvested and lipidomic analysis was performed using an ultra-high performance liquid chromatography-Orbitrap mass spectrometry system. Under the high altitude hypoxia environment, the spleen index of mice and the white pulp decreased, and the germinal center expanded with other pathological changes. The results of lipidomic analysis showed that a total of 41 lipid subclasses and 2 473 lipid molecules were identified, and triacylglycerides (TGs) and phosphatidylcholines (PCs) were the two most identified lipid molecules. Using univariate and multivariate analysis, 44 differentially expressed lipid molecules were identified, which were mainly concentrated in phospholipid metabolism. Subsequently, RT-qPCR was performed on the key enzymes hes in the phospholipid metabolic pathway, and it was found that the mRNA expression levels were different (P<0.05). It suggested that high altitude hypoxia environment mainly affects the phospholipid metabolism of mouse spleen tissue via reducing the contents of PC and phosphatidic acid (PA), promoting their conversion to phosphatidylethanolamine (PE) and cardiolipin (CL) and facilitating the PE production via the CDP-Etn pathway. This study provides a new experimental basis for the abnormal metabolism of phospholipids in spleen tissue under high altitude hypoxia environment. [ABSTRACT FROM AUTHOR]

Exosomes (Exo) are lipid-bilayer structures secreted by various cells, including those of animals, plants, and prokaryotes. Previous studies have revealed that Exo derived from humoral or cell-supernatant are promising targets for novel diagnostic or prognostic biomarkers, underscoring their significant role in disease pathogenesis. Tissue-derived Exo (Ti-Exo) have attracted increasing attention due to its ability to accurately reflect tissue specificity and the microenvironment. Ti-Exo, present in interstitial space, play crucial roles in intercellular communication and cross-organ signaling. Despite their recognized value in elucidating disease mechanisms, isolating Ti-Exo remains challenging due to the complexity of tissue matrices and variability in extraction methods. In this study, we developed a practical protocol for isolating exosomes from mice spleen tissue, providing a reproducible technique for subsequent identification analysis and functional studies. We used Type I collagenase digestion combined with differential ultracentrifugation to isolate spleen-derived Exo. The characteristics of isolated Exo were determined through electron microscopy, the nano-flow cytometer, and the western blot. The isolated spleen-derived Exo displayed the typical morphology of lipid bilayer vesicles, with particle sizes ranging from 30 nm to 150 nm. In addition, the expression profile of exosome markers confirmed the presence and purity of exosomes. Taken together, we successfully established a practical protocol for isolating spleen-derived Exo in mice.

High-altitude hypoxia exposure can lead to phospholipase D-mediated lipid metabolism disorder in spleen tissues and induce ferroptosis. Nonetheless, the key genes underlying hypoxia-induced splenic phospholipase D and the ferroptosis pathway remain unclear. This study aimed to establish a hypoxia animal model. Combined transcriptomic and proteomic analyses showed that 95 predicted target genes (proteins) were significantly differentially expressed under hypoxic conditions. Key genes in phospholipase D and ferroptosis pathways under hypoxic exposure were identified by combining Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis techniques. Gene set enrichment analysis (GSEA) showed that the differential gene sets of the phospholipase D and ferroptosis signaling pathways were upregulated in the high-altitude hypoxia group. The genes in the phospholipase D signalling pathway were verified, and the expression levels of KIT and DGKG were upregulated in spleen tissues under hypoxic exposure. Subsequently, the mRNA and protein expression levels of genes from the exogenous pathway such as TFRC, SLC40A1, SLC7A11, TRP53, and FTH1 and those from the endogenous pathway such as GPX4, HMOX1, and ALOX15 differentials in the ferroptosis signalling pathway were verified, and the results indicated significant differential expression. In summary, exposure to high-altitude hypoxia mediated phospholipid metabolism disturbance through the phospholipase D signalling pathway and further induced ferroptosis, leading to splenic injury.

In the context of the potential immunomodulatory properties of curcumin in counteracting the detrimental effects of concurrent exposure to Deoxynivalenol (DON) and Aflatoxin B1 (AFB1), a comprehensive 28-days trial was conducted utilizing 60 randomly allocated mice divided into four groups. Administration of curcumin at a dosage of 5 mg/kg body weight in conjunction with DON at 0.1 mg/kg and AFB1 at 0.01 mg/kg body weight was undertaken to assess its efficacy. Results indicated that curcumin intervention demonstrated mitigation of splenic structural damage, augmentation of serum immunoglobulin A (IgA) and immunoglobulin G (IgG) levels, elevation in T lymphocyte subset levels, and enhancement in the mRNA expression levels of pro-inflammatory cytokines TNF-α, IFN-γ, IL-2, and IL-6. Furthermore, curcumin exhibited a suppressive effect on apoptosis in mice, as evidenced by decreased activity of caspase-3 and caspase-9, reduced expression levels of pro-apoptotic markers Bax and Cytochrome-c (Cyt-c) at both the protein and mRNA levels, and the maintenance of a balanced expression ratio of mitochondrial apoptotic regulators Bax and Bcl-2. Collectively, these findings offer novel insights into the therapeutic promise of curcumin in mitigating immunosuppression and apoptotic events triggered by mycotoxin co-exposure.

In the context of the potential immunomodulatory properties of curcumin in counteracting the detrimental effects of concurrent exposure to Deoxynivalenol (DON) and Aflatoxin B1 (AFB1), a comprehensive 28-days trial was conducted utilizing 60 randomly allocated mice divided into four groups. Administration of curcumin at a dosage of 5 mg/kg body weight in conjunction with DON at 0.1 mg/kg and AFB 1 at 0.01 mg/kg body weight was undertaken to assess its efficacy. Results indicated that curcumin intervention demonstrated mitigation of splenic structural damage, augmentation of serum immunoglobulin A (IgA) and immunoglobulin G (IgG) levels, elevation in T lymphocyte subset levels, and enhancement in the mRNA expression levels of pro-inflammatory cytokines TNF-α, IFN-γ, IL-2, and IL-6. Furthermore, curcumin exhibited a suppressive effect on apoptosis in mice, as evidenced by decreased activity of caspase-3 and caspase-9, reduced expression levels of pro-apoptotic markers Bax and Cytochrome-c (Cyt-c) at both the protein and mRNA levels, and the maintenance of a balanced expression ratio of mitochondrial apoptotic regulators Bax and Bcl-2. Collectively, these findings offer novel insights into the therapeutic promise of curcumin in mitigating immunosuppression and apoptotic events triggered by mycotoxin co-exposure.

This study aims to investigate the antiallergic effects of Shiikuwasha (Citrus depressa Hayata) leaf and peel extracts by examining the regulation of degranulation and inflammatory cytokine production from rat basophilic leukemia (RBL-2H3) cells and antigen-specific antibody production in sensitized mouse spleen lymphocytes. In vivo antiallergic activity was evaluated using the passive cutaneous anaphylaxis (PCA) reaction model. Extracts of Shiikuwasha leaves and peel were prepared using 80% methanol and dissolved in dimethylsulfoxide. The dinitrophenyl-human serum albumin-induced β-hexosaminidase levels in immunoglobulin (Ig) E-sensitized RBL-2H3 cells were assessed using enzymatic assays. Cytokine production was measured by enzyme-linked immunosorbent assay. Antibody production capacity was evaluated using lymphocytes isolated from spleens of type I allergy model mice. Lymphocytes were cultured for 72 h with Shiikuwasha extracts, and ovalbumin-specific IgE, IgG1, and IgG2a levels were measured. Shiikuwasha leaf and peel extract significantly reduced β-hexosaminidase release and suppressed interleukin-4 and tumor necrosis factor-α production from RBL-2H3 cells. Ovalbumin-specific IgE and IgG1 production decreased in Shiikuwasha extract-treated lymphocytes. These extracts also significantly suppressed the PCA reaction. Shiikuwasha leaf and peel extract reduce degranulation in RBL-2H3 cells and antibody production in spleen-derived lymphocytes and therefore exhibit antiallergic effects.

Extramedullary erythropoiesis is not expected in healthy adult mice, but erythropoietic gene expression was elevated in lineage-depleted spleen cells from Cd47 -/- mice. Expression of several genes associated with early stages of erythropoiesis was elevated in mice lacking CD47 or its signaling ligand thrombospondin-1, consistent with previous evidence that this signaling pathway inhibits expression of multipotent stem cell transcription factors in spleen. In contrast, cells expressing markers of committed erythroid progenitors were more abundant in Cd47 -/- spleens but significantly depleted in Thbs1 -/- spleens. Single-cell transcriptome and flow cytometry analyses indicated that loss of CD47 is associated with accumulation and increased proliferation in spleen of Ter119 - CD34 + progenitors and Ter119 + CD34 - committed erythroid progenitors with elevated mRNA expression of Kit, Ermap, and Tfrc. Induction of committed erythroid precursors is consistent with the known function of CD47 to limit the phagocytic removal of aged erythrocytes. Conversely, loss of thrombospondin-1 delays the turnover of aged red blood cells, which may account for the suppression of committed erythroid precursors in Thbs1 -/- spleens relative to basal levels in wild-type mice. In addition to defining a role for CD47 to limit extramedullary erythropoiesis, these studies reveal a thrombospondin-1-dependent basal level of extramedullary erythropoiesis in adult mouse spleen., Competing Interests: RB, TM, MC, SK, DR No competing interests declared

This study aims to investigate the antiallergic effects of Shiikuwasha (Citrus depressa Hayata) leaf and peel extracts by examining the regulation of degranulation and inflammatory cytokine production from rat basophilic leukemia (RBL-2H3) cells and antigen-specific antibody production in sensitized mouse spleen lymphocytes. In vivo antiallergic activity was evaluated using the passive cutaneous anaphylaxis (PCA) reaction model. Extracts of Shiikuwasha leaves and peel were prepared using 80% methanol and dissolved in dimethylsulfoxide. The dinitrophenyl-human serum albumin-induced β-hexosaminidase levels in immunoglobulin (Ig) E-sensitized RBL-2H3 cells were assessed using enzymatic assays. Cytokine production was measured by enzyme-linked immunosorbent assay. Antibody production capacity was evaluated using lymphocytes isolated from spleens of type I allergy model mice. Lymphocytes were cultured for 72 h with Shiikuwasha extracts, and ovalbumin-specific IgE, IgG1, and IgG2a levels were measured. Shiikuwasha leaf and peel extract significantly reduced β-hexosaminidase release and suppressed interleukin-4 and tumor necrosis factor-a production from RBL-2H3 cells. Ovalbumin-specific IgE and IgG1 production decreased in Shiikuwasha extracttreated lymphocytes. These extracts also significantly suppressed the PCA reaction. Shiikuwasha leaf and peel extract reduce degranulation in RBL-2H3 cells and antibody production in spleen-derived lymphocytes and therefore exhibit antiallergic effects. [ABSTRACT FROM AUTHOR]

Cyclophosphamide (CP) is mainly used to treat autoimmune diseases and cancer; however, it damages normal immune cells. Therefore, the effects of chemotherapy on CP are limited. Notably, green tea has been reported to effectively modulate immune function. Here, given the pharmacological properties of green tea, we evaluated the ability of green tea extract (GTE) to restore immunity suppressed by CP in vivo and to activate macrophages in vitro. GTE significantly improved the suppressed immune function, including spleen index and proliferation of spleen T lymphocytes, as revealed by histopathological examination and flow cytometry analysis. Moreover, GTE effectively activated RAW 264.7, as represented by the induction of nitric oxide, reactive oxygen species, and cytokine levels. GTE also increased the phosphorylation of mitogen-activated protein kinases (MAPKs) and nuclear factor kappa B in RAW 264.7 cells. In conclusion, GTE ameliorated CP-induced immunosuppression in mice and stimulated immune activity in RAW 264.7 cells, possibly by activating the MAPK signaling pathway. These findings suggest that GTE has the potential to be used as a supplementary agent in chemotherapy for CP.

The objective: to study a response of the spleen extracellular matrix (ECM) to the liposome-encapsulated dextrazide (LEDZ) administration in the stabilization period of BCG-induced granulomatosis.Subjects and Methods. A model of generalized tuberculosis inflammation was reproduced by injecting mice with mycobacteria from BCG vaccine (BCG-infected mice). Intact mice served as controls. During inflammation stabilization, the infected mice were dosed with LEDZ by inhalation and intraperitoneal injection, and main ECM components were assessed.Results. The BCG-infected mice showed a decrease in the structural components of proteoglycans (PGs) but demonstrated elevated profibrotic fractions of hydroxyproline (Hyp), enhanced activity of matrix metalloproteinases (MMPs) and no differences in the content of their tissue inhibitors (TIMPs), that indicated the fibrosis of the organ. Despite the way of LEDZ administration, the level of uronic acids in PGs and activity of hyaluronidases increased but the content of protein-bound Hyp was reduced with no differences in the content of free Hyp versus infected untreated mice. The administration of LEDZ by inhalation reduced content of profibrotic fractions of Hyp but increased galactose in PGs increased to the level of control mice. The intraperitoneal LEDZ administration decreased the protein content in PGs.

Objective: To screen key differentially expressed genes （DEGs） in dead mice with sepsis by spleen high-throughput sequencing combined with bioinformatics. Methods: ①A mouse sepsis model was set up by intraperitoneal injection of lipopolysaccharide （LPS）, a 7-day survival curve of mice was drawn, and the modeling doses of survival group and death group were screened out. ②Expressions of TNF-α, IL-1β, IL-6 and IL-10 in peripheral blood of mice in control group, survival group and death group were verified by ELISA. ③High-throughput sequencing was conducted on spleens of survival group and death group, and the key genes were screened by bioinformatics analysis of DEGs. ④Expressions of key genes and proteins were detected by RT-PCR and Western blot. Results: ①LPS dosage in survival group was 15 mg/kg （with a mortality of 30%）, and LPS dosage in death group was 30 mg/kg（ with a mortality of 80%）. ②Expression levels of IL-6, TNF-α and IL-1β in sepsis mice were significantly higher than those of control group, while expression level of IL-10 was decreased （P<0.05）. Comparison of sepsis model groups showed that levels of pro-inflammatory factors in death group were higher than those in survival group, while level of IL-10 was lower than that in survival group （P<0.05）. ③A total of 2 999 DEGs in survival group and death group were screened out by bioinformatics, among which 1 185 genes were up-regulated and 1 814 genes were down-regulated. Top 5 DEGs enrichment pathways were screened out: "hematopoietic cell lineage" "primary immunodeficiency" "African trypanosomiasis" "leishmaniasis" and "B-cell receptor signaling pathway". Ifit1, Ifit3 and Mx1 were three key genes that were screened out. ④Compared with survival group, expressions of genes and proteins of Ifit1, Ifit3 and Mx1 were down-regulated in spleen tissues of the death group （P<0.05）. Conclusion: By high-throughput sequencing and bioinformatics, Ifit1, Ifit3 and Mx1 are screened out as key genes related to the death outcome of sepsis, which probably influence the outcome of sepsis through the immune mechanism related to virus infection. [ABSTRACT FROM AUTHOR]

Summary: Lymphoid tissue stromal cells are important regulators of spleen homeostasis and immune responses. Here, we present an optimized protocol that describes the digestion and enrichment steps for the isolation and analysis of rare populations of stromal cells, including fibroblastic reticular cells, perivascular cells, and glial cells found in the spleen. This protocol is suitable for subsequent analysis of spleen stromal cells by flow cytometry or single-cell RNA sequencing and to analyze different disease models.For complete details on the use and execution of this protocol, please refer to Alexandre et al. (2022).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Objective To investigate the direct effects of carboxyamidotriazole(CAI)on the survival and apoptosis of colon cancer cells(MC38 and C26)and its indirect effects through enhancing the anticancer effects of spleen lymphocytes in mice. Methods MC38 and C26 cells were cultured in vitro,spleen lymphocytes of mice were isolated,MC38 and C26 cells were co-cultured with spleen lymphocytes of mice,and the cells were treated with different concentrations of CAI. CCK-8 method was used to detect cell survival,flow cytometry was used to detect cell apoptosis and the proportion of CD4+CD3+ T cells and CD8+CD3+ T cells in spleen lymphocytes of mice. Results The number of living cells of MC38 and C26 cells decreased(P＜0.05,P＜0.01 and P＜0.001)and the apoptosis rate was increased(P＜0.01 and P＜0.001)after being treated with different concentrations of CAI. The number of living cells of MC38 and C26 cells was decreased(P＜0.001)upon being co-cultured with spleen lymphocytes of mice for 48 h, and the apoptosis rate was increased(P＜0.001). The number of living cells of MC38 and C26 cells in co- culture groups treated with different concentrations of CAI for 48 h further decreased(P＜0.001)and the apoptosis rate increased(P＜0.05 and P＜0.001)as compared with those co-cultured with spleen lymphocytes of mice without CAI. The proportion of CD4+CD3+ T cells and CD8+CD3+ T cells in spleen lymphocytes of mice was significantly increased(P＜0.05 and P＜0.01)after 48 h treatment with different concentrations of CAI. Conclusions CAI can inhibit the survival and promote apoptosis of MC38 and C26 cells,and enhance the effects of spleen lymphocytes in mice on inhibiting the survival and promoting apoptosis of MC38 and C26 cells through a potential mechanism of increasing the proportion of CD4+CD3+ T cells and CD8+CD3+ T cells in spleen lymphocytes of mice.

Transplantation of hematopoietic material into recipient mice is an assay routinely used to determine the presence and function of hematopoietic stem and progenitor cells (HSPCs) in vivo. The principle of the method is to transplant donor cells being tested for HSPCs into a recipient mouse following bone marrow ablation and testing for reconstitution of hematopoiesis. Congenic mouse strains where donor and recipient differ by a distinct cell surface antigen (commonly CD45.1 versus CD45.2) are used to distinguish between cells derived from the donor and any residual recipient cells. Typically, the transplantation is performed using bone marrow cells, which are enriched for HSPCs. Here, we describe an analogous procedure using hematopoietic material from spleen, allowing detection of functional progenitors and/or stem cells in the spleen that can occur under certain pathologies. Key to the success of this procedure is the prior removal of mature T cells from the donor sample, to minimize graft versus host reactions. As such, this protocol is highly analogous to standard bone marrow transplant procedures, differing mainly only in the source of stem cells (spleen rather than bone marrow) and the recommendation for T cell depletion to avoid potential immune incompatibilities.Graphical abstract: Schematic overview for assessment of stem cells in spleen by transplantation. Single cell suspensions from spleens are depleted of potentially pathogenic mature T lymphocytes by magnetic bead immunoselection using biotinylated antibodies against CD4 and CD8, followed by streptavidin magnetic beads, which are subsequently removed by using a magnet (MojoSort, Biolegend). Successful T cell depletion is then evaluated by Fluorescence Activated Cell Sorting (FACS). T-cell depleted cell suspension is injected intravenously through the retro-orbital sinus into lethally irradiated recipients. Recipients are analyzed for successful engraftment by FACS analysis for the presence of donor-derived mature hematopoietic lineages in the peripheral blood. A second serial transplantation can be used to document the presence of long-term reconstituting stem cells in the periphery of the original donor mice.

Carotenoids are the most abundant lipid-soluble phytochemicals and are used as dietary supplements to protect against diseases caused by oxidative stress. Astaxanthin, a xanthophyll carotenoid, is a very potent antioxidant with numerous beneficial effects on cellular functions and signaling pathways. In this study, using spleen cells from healthy Balb/c mice, we report the bio-functional effects of an astaxanthin-rich extract (EXT) prepared from the microalga Haematococcus pluvialis and its astaxanthin monoesters-rich fraction (ME) and astaxanthin diesters-rich fraction (DE) obtained by fractionation of EXT using countercurrent chromatography (CCC). After incubation under standard culture conditions (humidity, 37 °C, 5% CO2, atmospheric oxygen), the viability of untreated splenocytes, as determined by the trypan blue exclusion assay, the MTT assay, and the neutral red assay, decreases to approximately 75% after 24 h compared with naïve splenocytes. This effect correlated with the decrease in mitochondrial membrane potential and the transition of ~59% of cells to the early stage of apoptosis, as well as with the decreased ROS production, indicating that hyperoxia in cell-culture deteriorates cell functions. They are restored or stimulated by co-cultivation with EXT, ME, and DE up to 10 µg/mL in the order EXT > DE > ME, suggesting that esterification increases bioavailability to cells in vitro. ROS and H2O2 concentrations reflect mRNA transcriptional activity of Nrf2, superoxide dismutase 1 (SOD1), catalase, and glutathione peroxidase 1, as well as SOD-mediated ROS conversion, whereas they inversely correlate with iNOS-mediated NO production. The highest-tested concentration of EXT, ME, and DE (40 µg/mL) is detrimental to cells, probably because of the overwhelming scavenging activity of astaxanthin and its esters for the reactive oxygen/nitrogen species required for cellular functions and signal transduction at low physiological concentrations. In this study, we demonstrate that differential activities of ME and DE contribute to the final antioxidant and cytoprotective effects of astaxanthin extract, which is beneficial in preventing a wide range of ROS-induced adverse effects, with DE being more effective. In addition, the selection of physioxia-like conditions for pharmacological research is highlighted.

We have developed an approach to reprogramming immune cells by inhibiting the MAPK/ERK pathway through MEKi and the PD-1/PD-L1 immune checkpoint signaling pathway. We hypothesized that reprogramming of spleen CD8+ T-cells could also create a population of immune cells with high antitumor activity. We reprogrammed CD8+ T-cells derived from the spleen of C57BL/6 mice (rsCD8+T-cells). In the orthotopic LLC model, cell therapy with rsCD8+T-cells increased the amount of proliferating CD8+ and CD4+ T-cells in blood and lung tissue from mice. The amount of cancer stem cells (CSC) decreased in the blood and lung of mice treated with rsCD8+ T-cells. A morphological study revealed a decrease in the number of metastases in lung tissue. The antitumor effects of rsCD8+T-cells are based on the activation of the host immune response by increasing the populations of CD8+ and CD4+ T-cells and apoptosis of CSCs.