Immune Spleen Cells Research Articles

Dayuan Yin alleviates symptoms of HCoV-229E-induced pneumonia and modulates the Ras/Raf1/MEK/ERK pathway

Viral pneumonia is characterized by inflammation in the lungs triggered by respiratory viruses. Dayuan Yin (DYY), a traditional Chinese medicine formula known for treating infectious diseases, is hypothesized to offer therapeutic benefits in treating viral pneumonia, although its specific molecular impacts remain understudied. This study aimed to evaluate the therapeutic effects of DYY in mitigating HCoV-229E virus-induced pneumonia in mice. This study employed an HCoV-229E virus-infected mouse model to investigate the therapeutic potential and underlying molecular mechanisms of DYY on virus-induced pneumonia. The respiratory function and organ indices post-treatment were assessed. Lung tissue and tracheal lesions were evaluated via immunohistochemistry. Spleen immune cell composition was analyzed using flow cytometry. Inflammatory cytokines and viral loads were quantified using hypersensitive multiplex electrochemiluminescence method and PCR analysis, respectively. The expression levels of MAS1, Ras, Raf1, MEK1/2, and ERK1/2 in lung tissues were determined through western blot analysis. DYY significantly improved respiratory function, and reduced organ pathology in infected mice. It effectively decreased viral loads and inflammatory cytokines such as IL-6, IL-1β, and TNF-α in lung tissues. Enhancements in immune response were evidenced by increased CD4/CD8 ratios in the spleen. DYY also notably upregulated MAS1 protein levels and suppressed the activation of the Ras/Raf1/MEK/ERK signaling pathway. DYY enhanced respiratory function and exerted significant antiviral and immunomodulatory effects in mice infected with the HCoV-229E virus, primarily by modulating MAS1 expression and inhibiting the Ras/Raf1/MEK/ERK pathway.Graphical

Developmental Vitamin D Deficiency and the Vitamin D Receptor Control Hematopoiesis.

Vitamin D status, the vitamin D receptor (VDR), and the ability to produce active vitamin D [1,25(OH)2D, regulated by Cyp27b1] regulate fetal and adult hematopoiesis. Transgenic reporter mice that express the tdTomato RFP as an indication of Vdr expression were used to identify immune cells that express the Vdr. Vdr/tdTomato+ hematopoietic progenitors were identified as early as embryonic day (E)15.5, establishing that these cells have expressed the Vdr and are vitamin D targets. Maternal vitamin D deficiency [D-; serum 25(OH)D < 20 ng/ml] or Vdr knockout or Cyp27b1 knockout resulted in embryos with fewer fetal progenitors. Vdr/tdTomato+ expression was found to increase with age in CD8+ T cells and innate lymphoid cells (ILCs)1 and ILC3, suggesting that initial Vdr expression in these cells is dependent on environmental factors immediately postbirth. In adult tissues, the frequencies of mature T cells and ILCs as well as Vdr/tdTomato expression were reduced by D-. Maternal D- resulted in fewer progenitors that expressed Vdr/tdTomato+ at E15.5 and fewer Vdr/tdTomato+ immune cells in the adult spleen than offspring from D+ mice. We challenged D- mice with H1N1 influenza infection and found that D- mice were more susceptible than D+ mice. Treating D- mice with vitamin D restored Vdr/tdTomato+ expression in splenic T cells and partially restored resistance to H1N1 infection, which shows that developmental D- results in lingering effects on Vdr expression in the adult immune system that compromise the immune response to H1N1 infection. Vitamin D and the Vdr regulate hematopoiesis in both fetal and postnatal phases of immune cell development that impact the immune response to a viral infection.

Exposure to polystyrene microplastics during lactational period alters immune status in both male mice and their offspring

The widespread use of microplastics and their harmful effects on the environment have emerged as serious concerns. However, the effect of microplastics on the immune system of mammals, particularly their offspring, has received little attention. In this study, polystyrene microplastics (PS-MPs) were orally administered to male mice during lactation. Flow cytometry was used to assess the immune cells in the spleens of both adult male mice and their offspring. The results showed that mice exposed to PS-MPs exhibited an increase in spleen weight and an elevated number of B and regulatory T cells (Tregs), irrespective of dosage. Furthermore, the F1 male offspring of the PS-MPs-exposed group had enlarged spleens; an increased number of B cells, T helper cells (Th cells), and Tregs; and an elevated ratio of T helper cells 17 (Th17 cells) to Tregs and T helper cells 1 (Th1 cells) to T helper cells 2 (Th2 cells). These results suggested a pro-inflammatory state in the spleen. In contrast, in the F1 female offspring exposed to PS-MPs, the changes in splenic immune cells were less pronounced. In the F2 generation of mice with exposed to PS-MPs, minimal alterations were observed in spleen immune cells and morphology. In conclusion, our study demonstrated that exposure to real human doses of PS-MPs during lactation in male mice altered the immune status, which can be passed on to F1 offspring but is not inherited across generations.

Recombinant Hemagglutinin Protein from H9N2 Avian Influenza Virus Exerts Good Immune Effects in Mice.

The H9N2 subtype of avian influenza virus (AIV) causes enormous economic losses and poses a significant threat to public health; the development of vaccines against avian influenza is ongoing. To study the immunogenicity of hemagglutinin (HA) protein, we constructed a recombinant pET-32a-HA plasmid, induced HA protein expression with isopropyl β-D-1-thiogalactopyranoside (IPTG), verified it by SDS-PAGE and Western blotting, and determined the sensitivity of the recombinant protein to acid and heat. Subsequently, mice were immunized with the purified HA protein, and the immunization effect was evaluated according to the hemagglutination inhibition (HI) titer, serum IgG antibody titer, and cytokine secretion level of the mice. The results showed that the molecular weight of the HA protein was approximately 84 kDa, and the protein existed in both soluble and insoluble forms; in addition, the HA protein exhibited good acid and thermal stability, the HI antibody titer reached 6 log2-8 log2, and the IgG-binding antibody titer was 1:1,000,000. Moreover, the levels of IL-2, IL-4, and IL-5 in the immunized mouse spleen cells were significantly increased compared with those in the control group. However, the levels of IL-1β, IL-6, IL-13, IFN-γ, IL-18, TNF-α, and GM-CSF were decreased in the immunized group. The recombinant HA protein utilized in this study exhibited good stability and exerted beneficial immune effects, providing a theoretical basis for further research on influenza vaccines.

Sex-Dependent T Cell Dysregulation in Mice with Diet-Induced Obesity.

Obesity is an emerging public health problem. Chronic low-grade inflammation is considered a major promotor of obesity-induced secondary diseases such as cardiovascular and fatty liver disease, type 2 diabetes mellitus, and several cancer entities. Most preliminary studies on obesity-induced immune responses have been conducted in male rodents. Sex-specific differences between men and women in obesity-induced immune dysregulation have not yet been fully outlined but are highly relevant to optimizing prevention strategies for overweight-associated complications. In this study, we fed C57BL/6 female vs. male mice with either standard chow or an obesity-inducing diet (OD). Blood and spleen immune cells were isolated and analyzed by flow cytometry. Lean control mice showed no sex bias in systemic and splenic immune cell composition, whereas the immune responses to obesity were significantly distinct between female and male mice. While immune cell alterations in male OD mice were characterized by a significant reduction in T cells and an increase in myeloid-derived suppressor cells (MDSC), female OD mice displayed preserved T cell numbers. The sex-dependent differences in obesity-induced T cell dysregulation were associated with varying susceptibility to body weight gain and fatty liver disease: Male mice showed significantly more hepatic inflammation and histopathological stigmata of fatty liver in comparison to female OD mice. Our findings indicate that sex impacts susceptibility to obesity-induced T cell dysregulation, which might explain sex-dependent different incidences in the development of obesity-associated secondary diseases. These results provide novel insights into the understanding of obesity-induced chronic inflammation from a sex-specific perspective. Given that most nutrition, exercise, and therapeutic recommendations for the prevention of obesity-associated comorbidities do not differentiate between men and women, the data of this study are clinically relevant and should be taken into consideration in future trials and treatment strategies.

Infant Microbiota Communities and Human Milk Oligosaccharide Supplementation Independently and Synergistically Shape Metabolite Production and Immune Responses in Healthy Mice

BackgroundMultiple studies have demonstrated associations between the early-life gut microbiome and incidence of inflammatory and autoimmune disease in childhood. Although microbial colonization is necessary for proper immune education, it is not well understood at a mechanistic level how specific communities of bacteria promote immune maturation or drive immune dysfunction in infancy. ObjectivesIn this study, we aimed to assess whether infant microbial communities with different overall structures differentially influence immune and gastrointestinal development in healthy mice. MethodsGerm-free mice were inoculated with fecal slurries from Bifidobacterium longum subspecies infantis positive (BIP) or B. longum subspecies infantis negative (BIN) breastfed infants; half of the mice in each group were also supplemented with a pool of human milk oligosaccharides (HMOs) for 14 d. Cecal microbiome composition and metabolite production, systemic and mucosal immune outcomes, and intestinal morphology were assessed at the end of the study. ResultsThe results showed that inoculation with a BIP microbiome results in a remarkably distinct microbial community characterized by higher relative abundances of cecal Clostridium senu stricto, Ruminococcus gnavus, Cellulosilyticum sp., and Erysipelatoclostridium sp. The BIP microbiome produced 2-fold higher concentrations of cecal butyrate, promoted branched short-chain fatty acid (SCFA) production, and further modulated serotonin, kynurenine, and indole metabolism relative to BIN mice. Further, the BIP microbiome increased the proportions of innate and adaptive immune cells in spleen, while HMO supplementation increased proliferation of mesenteric lymph node cells to phorbol myristate acetate and lipopolysaccharide and increased serum IgA and IgG concentrations. ConclusionsDifferent microbiome compositions and HMO supplementation can modulate SCFA and tryptophan metabolism and innate and adaptive immunity in young, healthy mice, with potentially important implications for early childhood health.

Deciphering the impact of aging on splenic endothelial cell heterogeneity and immunosenescence through single-cell RNA sequencing analysis

BackgroundAging is associated with significant structural and functional changes in the spleen, leading to immunosenescence, yet the detailed effects on splenic vascular endothelial cells (ECs) and their immunomodulatory roles are not fully understood. In this study, a single-cell RNA (scRNA) atlas of EC transcriptomes from young and aged mouse spleens was constructed to reveal age-related molecular changes, including increased inflammation and reduced vascular development and also the potential interaction between splenic endothelial cells and immune cells.ResultsTen clusters of splenic endothelial cells were identified. DEGs analysis across different EC clusters revealed the molecular changes with aging, showing the increase in the overall inflammatory microenvironment and the loss in vascular development function of aged ECs. Notably, four EC clusters with immunological functions were identified, suggesting an Endothelial-to-Immune-like Cell Transition (EndICLT) potentially driven by aging. Pseudotime analysis of the Immunology4 cluster further indicated a possible aging-induced transitional state, potentially initiated by Ctss gene activation. Finally, the effects of aging on cell signaling communication between different EC clusters and immune cells were analyzed.ConclusionsThis comprehensive atlas elucidates the complex interplay between ECs and immune cells in the aging spleen, offering new insights into endothelial heterogeneity, reprogramming, and the mechanisms of immunosenescence.

Traumatic brain injury alters the effects of class II invariant peptide (CLIP) antagonism on chronic meningeal CLIP + B cells, neuropathology, and neurobehavioral impairment in 5xFAD mice

BackgroundTraumatic brain injury (TBI) is a significant risk factor for Alzheimer’s disease (AD), and accumulating evidence supports a role for adaptive immune B and T cells in both TBI and AD pathogenesis. We previously identified B cell and major histocompatibility complex class II (MHCII)-associated invariant chain peptide (CLIP)-positive B cell expansion after TBI. We also showed that antagonizing CLIP binding to the antigen presenting groove of MHCII after TBI acutely reduced CLIP + splenic B cells and was neuroprotective. The current study investigated the chronic effects of antagonizing CLIP in the 5xFAD Alzheimer’s mouse model, with and without TBI.Methods12-week-old male wild type (WT) and 5xFAD mice were administered either CLIP antagonist peptide (CAP) or vehicle, once at 30 min after either sham or a lateral fluid percussion injury (FPI). Analyses included flow cytometric analysis of immune cells in dural meninges and spleen, histopathological analysis of the brain, magnetic resonance diffusion tensor imaging, cerebrovascular analysis, and assessment of motor and neurobehavioral function over the ensuing 6 months.Results9-month-old 5xFAD mice had significantly more CLIP + B cells in the meninges compared to age-matched WT mice. A one-time treatment with CAP significantly reduced this population in 5xFAD mice. Importantly, CAP also improved some of the immune, histopathological, and neurobehavioral impairments in 5xFAD mice over the ensuing six months. Although FPI did not further elevate meningeal CLIP + B cells, it did negate the ability of CAP to reduce meningeal CLIP + B cells in the 5xFAD mice. FPI at 3 months of age exacerbated some aspects of AD pathology in 5xFAD mice, including further reducing hippocampal neurogenesis, increasing plaque deposition in CA3, altering microgliosis, and disrupting the cerebrovascular structure. CAP treatment after injury ameliorated some but not all of these FPI effects.

Pathological changes in the spleen of mice subjected to different time courses of restraint stress

The objective of this study was to investigate spleen pathology and immune cell subset alterations in mice exposed to acute and chronic restraint stress over various timeframes. A deeper understanding of stress-induced spleen injuries can provide new insights into the mechanisms underlying stress-induced disorders. C57BL/6N mice were restrained for different durations (1, 3, 7, 14 and 21 days) for 6–8 h daily. The control mice were observed at the same time points. Post restraint, behavioural experiments were conducted to assess spleen weight, gross morphology and microscopic histological changes. Immunohistochemical staining was used to detect changes in glucocorticoid receptor (GR) expression, immune cell subsets and cell proliferation in response to stress. Our analysis revealed significant behavioural abnormalities in the stressed mice. In particular, there was an increase in the nuclear expression of GR beginning on Day 3, and it peaked on Day 14. The spleens of stressed mice displayed a reduction in size, disordered internal tissue structure and reduced cell proliferation. NK cells and M2-type macrophages exhibited immune cell subset alterations under stress, whereas T or B cells remained unaltered. Restraint stress can lead to pathomorphological alterations in spleen morphology, cell proliferation and immune cell counts in mice. These findings suggest that stress-induced pathological changes can disrupt immune regulation during stress.

Astragalus polysaccharides attenuate chemotherapy-induced immune injury by modulating gut microbiota and polyunsaturated fatty acid metabolism

BackgroundThe damage of chemotherapy drugs to immune function and intestinal mucosa is a common side effect during chemotherapy. Astragalus polysaccharides (APS) exhibit immunomodulatory properties and are recognized for preserving the integrity of the human intestinal barrier. Nevertheless, their application and mechanisms of action in chemotherapy-induced immune damage and intestinal barrier disruption remain insufficiently explored. PurposeThis study delved into investigating how APS mitigates chemotherapy-induced immune dysfunction and intestinal mucosal injury, while also providing deeper insights into the underlying mechanisms. MethodsIn a chemotherapy mice model induced by 5-fluorouracil (5-Fu), the assessment of APS's efficacy encompassed evaluations of immune organ weight, body weight, colon length, and histopathology. The regulation of different immune cells in spleen was detected by flow cytometry. 16S rRNA gene sequencings, ex vivo microbiome assay, fecal microbiota transplantation (FMT), and targeted metabolomics analysis were applied to explore the mechanisms of APS effected on chemotherapy-induced mice. ResultsAPS ameliorated chemotherapy-induced damage to immune organs and regulated immune cell differentiation disorders, including CD4+T, CD8+T, CD19+B, F4/80+CD11B+ macrophages. APS also alleviated colon shortening and upregulated the expression of intestinal barrier proteins. Furthermore, APS significantly restored structure of gut microbiota following chemotherapy intervention. Ex vivo microbiome assays further demonstrated the capacity of APS to improve 5-Fu-induced microbiota growth inhibition and compositional change. FMT demonstrated that the regulation of gut microbiota by APS could promote the recovery of immune functions and alleviate shortening of the colon length. Remarkably, APS significantly ameliorated the imbalance of linoleic acid (LA) and α-linolenic acid in polyunsaturated fatty acid (PUFA) metabolism. Further in vitro experiments showed that LA could promote splenic lymphocyte proliferation. In addition, both LA and DGLA down-regulated the secretion of NO and partially up-regulated the percentage of F4/80+CD11B+CD206+ cells. ConclusionAPS can effectively ameliorate chemotherapy-induced immune damage and intestinal mucosal disruption by regulating the composition of the gut microbiota and further restoring PUFA metabolism. These findings indicate that APS can serve as an adjuvant to improve the side effects such as intestinal and immune damage caused by chemotherapy.

Deucravacitinib and shikonin combination therapy ameliorates imiquimod-induced psoriasis in mice.

TYK2 inhibitors and traditional natural drugs as promising drugs for psoriasis therapy are receiving increasing attention. They both affect different molecules of JAK/STAT pathway, but it is currently unclear whether their combination will enhance the effect on psoriasis. In this study, we used imiquimod (IMQ)-induced psoriasis mouse model to investigate the therapeutic effects of the combined administration of deucravacitinib (TYK2 inhibitor) and shikonin. Aldara cream containing 5% IMQ was used to topically treat the dorsal skin of each mouse for a total of six consecutive days to induce psoriasis. The psoriasis area and severity index (PASI) scores were recorded every day. On the 7th day, skin tissues were taken for histopathological examination and the content of cytokines in skin were evaluated. The frequency of immune cells in peripheral blood, spleen and skin were detected through flow cytometry. Compared to the vehicle control group, the psoriasis symptoms and immune disorder improved significantly in the combination therapy group and deucravacitinib treatment group on the 7th day, and the expressions of p-STAT3 and Ki67 in skin were reduced as well. Moreover, the combined treatment of deucravacitinib and shikonin for psoriasis was superior to the monotherapy group, especially in inhibiting abnormal capillaries proliferation, reducing immune cells infiltration and decreasing the concentration of IL-12p70 in skin. The combination of deucravacitinib and shikonin is a promising clinical application.

Kisspeptin prevents pregnancy loss by modulating the immune microenvironment at the maternal-fetal interface in recurrent spontaneous abortion.

Immune factors are crucial in the development of recurrent spontaneous abortion (RSA). This study aimed to investigate whether kisspeptin regulates immune cells at the maternal-fetal interface and whether G protein-coupled receptor 54 (GPR54) is involved in this process, through which it contributes to the pathogenesis of RSA. Normal pregnancy (NP) (CBA/J × BALB/c) and RSA (CBA/J × DBA/2) mouse models were established. NP mice received tail vein injections of PBS and KP234 (blocker of kisspeptin receptor), whereas RSA mice received PBS and KP10 (active fragment of kisspeptin). The changes in immune cells in mouse spleen and uterus were assessed using flow cytometry and immunofluorescence. The expression of critical cytokines was examined by flow cytometry, ELISA, Western blotting, and qPCR. Immunofluorescence was employed to detect the coexpression of FOXP3 and GPR54. The findings revealed that the proportion of Treg cells, MDSCs, and M2 macrophages in RSA mice was lower than that in NP mice, but it increased following the tail vein injection of KP10. Conversely, the proportion of these cells was reduced in NP mice after the injection of KP234. However, the trend of γδT cell proportion change is contrary to these cells. Furthermore, FOXP3 and GPR54 were coexpressed in mouse spleen and uterus Treg cells as well as in the human decidua samples. Our results suggest that kisspeptin potentially participates in the pathogenesis of RSA by influencing immune cell subsets at the maternal-fetal interface, including Treg cells, MDSC cells, γδT cells, and M2 macrophages.

STERILE NEUTROPHILIA INDUCED VIA CXCR4 ANTAGONISM MITIGATES COLONIC INFLAMMATION BY ENHANCING IMMUNOSUPPRESSIVE REGULATORY T CELLS IN MICE

Abstract Sterile neutrophilia (SN) is commonly associated with chronic diseases including inflammatory bowel diseases (IBD), but its defined role is largely unknown. Plerixafor (AMD3100) is a CXCR4 antagonist that promotes neutrophil egress from bone marrow resulting in SN. We first confirmed that C57BL6/J mice administered Plerixafor (10 mg/kg/bw, i.p.,) increased their circulating neutrophils by 50% within 2.5 h. Further, to study the role of SN in IBD, we induced chronic colitis by neutralizing IL-10 signaling via αIL-10R mAb (1.0 mg/mouse, i.p., 4 weekly injections) with or without weekly twice Plerixafor. Control mice were given isotype control mAb. As expected, αIL-10R+Plerixafor-treated mice had increased peripheral and splenic neutrophils. Further, these mice displayed remarkably reduced epithelial damage, hyperplasia, immune cell infiltration, and disease activity index when compared to αIL-10R+vehicle treated mice, which had typical colitis symptoms. To delineate the mechanism for such protection by Plerixafor, we profiled immune cells in circulation, spleen and lamina propria. In circulation αIL-10R+Plerixafor-treated mice had elevated CD3+CD4+ helper T cells (Th) but reduced CD3+CD8+ cytotoxic T (Tc) cells and CD3+CD4+FoxP3+ cells (Tregs) and CD3+CD4+RORγt+ cells (Th17) remained unchanged. While in the spleens of αIL-10R+Plerixafor-treated mice displayed significant increased Tregs, but decreased Th and Tc cells. Interestingly, markedly elevated Tregs, RORγt+ Tregs and tolerogenic IL-10+ Tregs was observed in lamina propria when compared to αIL-10R+ vehicle given mice. Interestingly, the proinflammatory IL-17+ Th cells were decreased in αIL-10R+Plerixafor-treated mice which showed mild colitis. Moreover, to further elucidate the role of Plerixafor in intestinal barrier function in colitic mice, we noticed treatment with Plerixafor increased the expression of occludin and claudins (1, 2), which are the principal transmembrane tight junctions (TJ) proteins; however Plerixafor treatment reduced the expression of zonula occludens (ZO), a peripheral membrane TJ protein. Collectively, our results demonstrate that SN could be an adaptive and beneficial immune response during chronic intestinal inflammation.

Maternal consumption of a high-fat diet modulates the inflammatory response in their offspring, mediated by the M1 muscarinic receptor.

High-fat diet (HFD) consumption is associated with various metabolic disorders and diseases. Both pre-pregnancy and maternal obesity can have long-term consequences on offspring health. Furthermore, consuming an HFD in adulthood significantly increases the risk of obesity and metabolic disorders. However, an intriguing phenomenon known as the obesity paradox suggests that obesity may confer a protective effect on mortality outcomes in sepsis. In sepsis, activation of the cholinergic anti-inflammatory pathway (CAP) can help mitigate systemic inflammation. We employed a metabolic programming model to explore the relationship between maternal HFD consumption and offspring response to sepsis. We fed female mice either a standard diet (SC) or an HFD during the pre-pregnancy, pregnancy, and lactation periods. Subsequently, we evaluated 28-day-old male offspring. Notably, we discovered that offspring from HFD-fed dams (HFD-O) exhibited a higher survival rate compared with offspring from SC-fed dams (SC-O). Importantly, inhibition of the m1 muscarinic acetylcholine receptor (m1mAChR), involved in the CAP, in the hypothalamus abolished this protection. The expression of m1mAChR in the hypothalamus was higher in HFD-O at different ages, peaking on day 28. Treatment with an m1mAChR agonist could modulate the inflammatory response in peripheral tissues. Specifically, CAP activation was greater in the liver of HFD-O following agonist treatment. Interestingly, lipopolysaccharide (LPS) challenge failed to induce a more inflammatory state in HFD-O, in contrast to SC-O, and agonist treatment had no additional effect. Analysis of spleen immune cells revealed a distinct phenotype in HFD-O, characterized by elevated levels of CD4+ lymphocytes rather than CD8+ lymphocytes. Moreover, basal Il17 messenger RNA (mRNA) levels were lower while Il22 mRNA levels were higher in HFD-O, and we observed the same pattern after LPS challenge. Further examination of myeloid cells isolated from bone marrow and allowed to differentiate showed that HFD-O macrophages displayed an anti-inflammatory phenotype. Additionally, treatment with the m1mAChR agonist contributed to reducing inflammatory marker levels in both groups. In summary, our findings demonstrate that HFD-O are protected against LPS-induced sepsis, and this protection is mediated by the central m1mAChR. Moreover, the inflammatory response in the liver, spleen, and bone marrow-differentiated macrophages is diminished. However, more extensive analysis is necessary to elucidate the specific mechanisms by which m1mAChR modulates the immune response during sepsis.

In vitro and in vivo killing effects of methionine enkephalin on osteosarcoma

ObjectiveThis study aimed to investigate the underlying regulatory effects of methionine enkephalin (MENK) on osteosarcoma. MethodsThe Cell Counting Kit-8 assay, clone formation, wound healing, transwell assay, and flow cytometry were performed to measure the effects of MENK on the proliferation, migration, invasion, and apoptosis of MG-63 and Saos-2 cells. Opiate growth factor receptor expression (OGFr) in cells was stably knocked down using siRNA. A tumor model was established by inoculating MG-63 cells into mice. Flow cytometry was performed to identify alterations in mice bone marrow, spleen, and tumor tissue immune cells. The phenotype of tumor-associated macrophages was determined using immunohistochemistry. After OGFr knockdown or/and treatment with MENK, Bax, Bcl-2, caspase 3, caspase 9, and PARP expression levels were characterized using qRT-PCR, western blot, and WES, respectively. ResultsMENK could significantly inhibit the proliferation, invasion, and migration of MG-63 and Saos-2, arrest the cell cycle in the G0/G1 phase, upregulate Bax, caspase 3, caspase 9, and PARP expression, and downregulate Bcl-2 expression. Tumor size and weight were lower in the MENK group than those in the control group. MENK-treated mice exhibited a reduced ratio of CD11b + Gr-1 + myeloid-derived suppressor cells. MENK increased the ratio of M1-type macrophages and decreased the proportion of M2-type macrophages in tumor tissue. Furthermore, the level of TNF-α significantly increased while that of IL-10 decreased in MENK-treated mice. The effect of MENK could be partly reversed by OGFr knockdown. ConclusionMENK reduces the abundance of myeloid-derived suppressor cells, induces M1 polarization of macrophages, and exhibits an inhibitory effect on osteosarcoma.

Effects of Systemic Peroxisome Proliferator-Activated Receptor Gamma Inhibition on Bone and Immune Cells in Aged Female Mice.

This study investigates the effects of peroxisome proliferator-activated receptor gamma (PPARγ) inhibition on bone and immune cell profiles in aged female mice, as well as in vitro stromal stem cell osteogenic differentiation and inflammation gene expression. The hypothesis was that inhibition of PPARγ would increase bone mass and alter immune and other cellular functions. Our results showed that treatment with PPARγ antagonist GW9662 for 6 weeks reduced bone volume and trabecular number and increased trabecular spacing. However, inhibition of PPARγ had no significant effect on marrow and spleen immune cell composition in aged female mice. In vitro experiments indicated that GW9662 treatment increased the expression of osteogenic genes but did not affect adipogenic genes. Additionally, GW9662 treatment decreased the expression of several inflammation-related genes. Overall, these findings suggest that PPARγ inhibition may have adverse effects on bone in aged female mice.

High-fat diet alters immune cells in spleen, kidney and tumor and impacts the volume growth of renal cell carcinoma

Renal cell carcinoma (RCC) is strongly associated with abnormal or excessive fat deposition in the body, whose processes include persistent adipose inflammation and other disturbances with the development and function of immune cells. Researchers have recently become more and more interested in understanding how high-fat diet (HFD) affects the development and course of RCC by causing immunological dysfunction. Consequently, we explore the effect of HFD on the changes of immune cell groups in spleens, normal kidneys and tumors, mainly revealing the changes of T cells, B cells and NK cells, and further preliminarily exploring the changes of NK cell phenotype. Our findings demonstrate that: (1) HFD impacts the volume growth of ACHN tumor; (2) HFD increases the frequency of CD3+ T cell in spleen, normal kidney, and in tumor, while there are no significant change in CD19+ B cell in spleen, normal kidney and tumor; (3) HFD increases the frequency of NKp46+ NK cell in tumor, while HFD decrease the frequency of NKp46+ NK cell in spleen; (4) HFD increases the frequency of cNK in spleen, normal kidney and tumor, while HFD decreases the frequency of ILC1 in spleen, normal kidney and tumor. These data will open up new avenues for immunotherapy in individuals with obese renal cell carcinoma.

Abstract P3097: Sex-related Differences In Immune Responsiveness Of Sarcospan-deficient Mice To Metabolic Stress

This study compares sex-related differences in inflammatory signaling that may contribute to metabolic and cardiovascular disease. The SSPN gene encoding the sarcospan (SSPN) protein has been described as an “obesity-susceptibility” gene. To study the influence of SSPN on metabolic function we subjected young 2-month-old male and female WT and sarcospan-deficient (SSPN -/- ) mice to 4 months high fat diet (HFD) (60% fat). Young male and female SSPN -/- mice exhibited some protection from weight gain and tissue inflammation. Cardiac function was preserved in the SSPN -/- mice of both sexes after HFD. Thus, we became interested in the combined impact of SSPN and metabolic stress on systemic inflammation. To study how metabolic stress impacts activation of anti- and pro-inflammatory pathways the chemokine/cytokine/adipokine proteome was examined in blood serum, heart, white adipose tissue, and liver of male mice. Under control conditions both WT and SSPN -/- male blood serum exhibited low levels of pro-inflammatory molecules, however CXCL 10 and CCL-2 significantly increased in SSPN -/- after HFD. Histological analysis of white adipose tissue (WAT) further revealed that SSPN deficiency may be protective against WAT immune infiltration in both males and females. Proteome analysis of WAT from HFD and control diet males revealed striking alterations in inflammatory signatures, whereas in the heart and liver most significant changes were observed in SSPN -/- controls. Flow cytometry was performed to examine spleen immune cell content of the mice. To elucidate factors contributing to the sex differences observed in the SSPN -/- response to metabolic stress we examined the response of bone marrow derived macrophages (BMDM) obtained from the sex/diet/gender groups included in this study. The BMDM acute response to innate immune ligands was tested at 3 and 6 hours to innate immune ligands and in general, female control diet SSPN -/- BMDMs exhibited enhanced pro-inflammatory responsiveness compared to female HFD and male control diet BMDMs. Overall, these studies describe distinct inflammatory signatures in the heart and WAT and a sex-dependent influence of SSPN -/- on macrophage activation that may have distinct consequences on cardiovascular and metabolic health.

Harnessing Topology and Stereochemistry of Glycidylamine‐Derived Lipid Nanoparticles for in Vivo mRNA Delivery to Immune Cells in Spleen and Their Application for Cancer Vaccination

AbstractmRNA lipid nanoparticles (LNPs) have reached an inflection point and are now paving the way for a new wave of precision therapies. The design of nonhepatocyte RNA delivery systems without targeting ligands, however, remains a challenge. It is reported that the development of ligand‐free glycidylamine (GA) derivatives containing LNPs (GA‐LNPs) that preferentially deliver mRNA to immune cells in the spleen. Notably, it is demonstrated that the stereochemistry of GA‐lipids has a significant impact on their self‐assembly and in vitro and in vivo RNA delivery efficiency and tropism. This impact is dependent on the monomeric structure of GA and number of stereogenic centers. Furthermore, the nonlinear topology of GA lipid derivatives induced a sevenfold improvement in mRNA delivery efficiency. The top‐performing estriol‐GA05‐30 LNPs elicited strong antitumor activity in a therapeutic and prophylactic cancer model and are well tolerated in mice. These results highlight the significance of the chemistry of ionizable lipids for extrahepatic RNA delivery and indicated a promising direction for the development of next‐generation mRNA immunotherapies.

Microbiota-mediated shaping of mouse spleen structure and immune function characterized by scRNA-seq and Stereo-seq

Gut microbes exhibit complex interactions with their hosts and shape an organism's immune system throughout its lifespan. As the largest secondary lymphoid organ, the spleen has a wide range of immunological functions. To explore the role of microbiota in regulating and shaping the spleen, we employ scRNA-seq and Stereo-seq technologies based on germ-free (GF) mice to detect differences in tissue size, anatomical structure, cell types, functions, and spatial molecular characteristics. We identify 18 cell types, 9 subtypes of T cells, and 7 subtypes of B cells. Gene differential expression analysis reveals that the absence of microorganisms results in alterations in erythropoiesis within the red pulp region and congenital immune deficiency in the white pulp region. Stereo-seq results demonstrate a clear hierarchy of immune cells in the spleen, including marginal zone (MZ) macrophages, MZ B cells, follicular B cells and T cells, distributed in a well-defined pattern from outside to inside. However, this hierarchical structure is disturbed in GF mice. Ccr7 and Cxcl13 chemokines are specifically expressed in the spatial locations of T cells and B cells, respectively. We speculate that the microbiota may mediate the structural composition or partitioning of spleen immune cells by modulating the expression levels of chemokines.