Brain control of humoral immune responses amenable to behavioural modulation.

It has been speculated that brain activities might directly control adaptive immune responses in lymphoid organs, although there is little evidence for this. Here we show that splenic denervation in mice specifically compromises the formation of plasma cells during a T cell-dependent but not T cell-independent immune response. Splenic nerve activity enhances plasma cell production in a manner that requires B-cell responsiveness to acetylcholine mediated by the α9 nicotinic receptor, and T cells that express choline acetyl transferase1,2 probably act as a relay between the noradrenergic nerve and acetylcholine-responding B cells. We show that neurons in the central nucleus of the amygdala (CeA) and the paraventricular nucleus (PVN) that express corticotropin-releasing hormone (CRH) are connected to the splenic nerve; ablation or pharmacogenetic inhibition of these neurons reduces plasma cell formation, whereas pharmacogenetic activation of these neurons increases plasma cell abundance after immunization. In a newly developed behaviour regimen, mice are made to stand on an elevated platform, leading to activation of CeA and PVN CRH neurons and increased plasma cell formation. In immunized mice, the elevated platform regimen induces an increase in antigen-specific IgG antibodies in a manner that depends on CRH neurons in the CeA and PVN, an intact splenic nerve, and B cell expression of the α9 acetylcholine receptor. By identifying a specific brain-spleen neural connection that autonomically enhances humoral responses and demonstrating immune stimulation by a bodily behaviour, our study reveals brain control of adaptive immunity and suggests the possibility to enhance immunocompetency by behavioural intervention.

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.

Plant- and Animal-Derived Dietary Sources of Phosphatidylcholine Have Differential Effects on Immune Function in The Context of A High-Fat Diet in Male Wistar Rats.

BACKGROUND: Phosphatidylcholine (PC) derived from eggs has been shown to beneficially modulate T cell response and intestinal permeability under the context of a high-fat diet. OBJECTIVES: The objective of this study was to determine whether there is a differential effect of plant and animal-derived sources of PC on immune function. METHODS: Four-week-old male Wistar rats were randomly assigned to consume 1 of 4 diets (n = 10/group) for 12 wk, all containing 1.5 g of total choline/kg of diet but differing in choline forms: 1-Control Low-Fat [CLF, 20% fat, 100% free choline (FC)]; 2-Control High-Fat (CHF, 50% fat, 100% FC); 3-High-Fat Egg-derived PC (EPC, 50% fat, 100% Egg-PC); 4-High-Fat Soy-derived PC (SPC, 50% fat, 100% Soy-PC). Immune cell functions and phenotypes were measured in splenocytes by ex vivo cytokine production after mitogen stimulation and flow cytometry, respectively. RESULTS: The SPC diet increased splenocyte IL-2 production after PMA+I stimulation compared with the CHF diet. However, the SPC group had a lower proportion of splenocytes expressing the IL-2 receptor (CD25+, P < 0.05). After PMA+I stimulation, feeding EPC normalized splenocyte production of IL-10 relative to the CLF diet, whereas SPC did not (P < 0.05). In mesenteric lymph node lymphocytes, the SPC diet group produced more IL-2 and TNF-α after PMA+I stimulation than the CHF diet, whereas the EPC diet group did not. CONCLUSIONS: Our results suggest that both egg- and soy-derived PC may attenuate high-fat diet-induced T cell dysfunction. However, egg-PC enhances, to a greater extent, IL-10, a cytokine involved in promoting the resolution phase of inflammation, whereas soy-PC appears to elicit a greater effect on gut-associated immune responses.

Diminazene aceturate attenuates systemic inflammation via microbiota gut-5-HT brain-spleen sympathetic axis in male mice.

The sympathetic arm of the inflammatory reflex is the efferent pathway through which the central nervous system (CNS) can control peripheral immune responses. Diminazene aceturate (DIZE) is an antiparasitic drug that has been reported to exert protective effects on various experimental models of inflammation. However, the pathways by which DIZE promotes a protective immunomodulatory effects still need to be well established, and no studies demonstrate the capacity of DIZE to modulate a neural reflex to control inflammation. C57BL/6 male mice received intraperitoneal administration of DIZE (2 mg/Kg) followed by lipopolysaccharide (LPS, 5 mg/Kg, i.p.). Endotoxemic animals showed hyperresponsiveness to inflammatory signals, while those treated with DIZE promoted the activation of the inflammatory reflex to attenuate the inflammatory response during endotoxemia. The unilateral cervical vagotomy did not affect the anti-inflammatory effect of DIZE in the spleen and serum. At the same time, splenic denervation attenuated tumor necrosis factor (TNF) synthesis in the spleen and serum. Using broad-spectrum antibiotics for two weeks showed that LPS modulated the microbiota to induce a pro-inflammatory profile in the intestine and reduced the serum concentration of tryptophan and serotonin (5-HT), while DIZE restored serum tryptophan and increased the hypothalamic 5-HT levels. Furthermore, the treatment with 4-Chloro-DL-phenylalanine (pcpa, an inhibitor of 5-HT synthesis) abolished the anti-inflammatory effects of the DIZE in the spleen. Our results indicate that DIZE promotes microbiota modulation to increase central 5-HT levels and activates the efferent sympathetic arm of the inflammatory reflex to control splenic TNF production in endotoxemic mice.

Immunostimulatory Effects of Korean Mineral-Rich Seawaters on Cyclophosphamide-Induced Immunosuppression in Mice.

Deep seawater (DS), obtained from a depth over 200 m, has health benefits due to its rich nutrients and minerals, and intake of DS has shown diverse immunomodulatory effects in allergies and cancer. Therefore, the immunostimulatory effects of Korean mineral-rich seawaters were examined in a cyclophosphamide (CPA)-induced immunosuppression model. Three samples of Korean seawater, namely DS from the East Sea off the coasts of Pohang (PDS) and Uljin (UDS), and seawater from the West Sea off the coast of Boryeong (BS), were collected. The seawaters were abundant in several minerals (calcium, iron, zinc, selenium, etc.). Mice were orally administered the seawaters for 42 days, followed by CPA-induced immunosuppression. The CPA induction reduced the weight of the spleen and lymph nodes; however, the administration of seawaters increased the weight of the lymphoid organs, accompanied by stimulation of natural killer cells' activity and NF-kB-mediated cytokine production (IFNγ, TNFα, IL1ß, IL6, and IL12). The mouse-derived splenocytes showed lymphoproliferation without cytotoxicity in the seawater groups. Histopathological analysis revealed that the seawaters improved the CPA-induced atrophic changes by promoting lymphoproliferation in the spleen and lymph nodes. These results provide useful information for the use of Korean mineral-rich seawaters, particularly PDS and UDS, as alternative immunostimulants under immunosuppressive conditions.

Human-relevant near-organ neuromodulation of the immune system via the splenic nerve.

Neuromodulation of immune function by stimulating the autonomic connections to the spleen has been demonstrated in rodent models. Consequently, neuroimmune modulation has been proposed as a new therapeutic strategy for the treatment of inflammatory conditions. However, demonstration of the translation of these immunomodulatory mechanisms in anatomically and physiologically relevant models is still lacking. Additionally, translational models are required to identify stimulation parameters that can be transferred to clinical applications of bioelectronic medicines. Here, we performed neuroanatomical and functional comparison of the mouse, rat, pig, and human splenic nerve using in vivo and ex vivo preparations. The pig was identified as a more suitable model of the human splenic innervation. Using functional electrophysiology, we developed a clinically relevant marker of splenic nerve engagement through stimulation-dependent reversible reduction in local blood flow. Translation of immunomodulatory mechanisms were then assessed using pig splenocytes and two models of acute inflammation in anesthetized pigs. The pig splenic nerve was shown to locally release noradrenaline upon stimulation, which was able to modulate cytokine production by pig splenocytes. Splenic nerve stimulation was found to promote cardiovascular protection as well as cytokine modulation in a high- and a low-dose lipopolysaccharide model, respectively. Importantly, splenic nerve-induced cytokine modulation was reproduced by stimulating the efferent trunk of the cervical vagus nerve. This work demonstrates that immune responses can be modulated by stimulation of spleen-targeted autonomic nerves in translational species and identifies splenic nerve stimulation parameters and biomarkers that are directly applicable to humans due to anatomical and electrophysiological similarities.

Study on the active components and mechanism of Atractylodis Macrocephalae Rhizoma for invigorating the spleen and tonifying qi based on spectrum-effect relationship and network pharmacology.

Spleen deficiency can lead to various abnormal physiological functions of the spleen. Atractylodis Macrocephalae Rhizoma (AMR) is a traditional Chinese medicine used to invigorate the spleen and tonify qi. The study aimed to identify the primary active components influencing the efficacy of AMR in strengthening the spleen and replenishing qi through spectrum-effect relationship and chemometrics. Network pharmacology was used to investigate the mechanism by which AMR strengthens the spleen and replenishes qi, with molecular docking utilized for validation purposes. The findings indicated that bran-fried AMR exhibited superior efficacy, with atractylenolides and atractylone identified as the primary active constituents. Atractylenolide II emerged as the most influential component impacting the effectiveness of AMR, while the key target was androgen receptor. Furthermore, crucial pathways implicated included the mitogen-activated protein cascade (MAPK) cascade, RNA polymerase II transcription factor activity, ligand-activated sequence-specific DNA binding, and RNA polymerase II sequence-specific DNA-binding transcription factor binding. In summary, our study has identified the primary active components associated with the efficacy of AMR and has provided an initial exploration of its mechanism of action. This offers a theoretical foundation for future investigations into the material basis and molecular mechanisms underlying the pharmacodynamics of AMR.

Cadmium promoted LPS-induced inflammation through TLR4/IκBα/NFκ-B signaling by increasing ROS-mediated incomplete autophagy.

Cadmium (Cd) exposure is considered as non-infectious stressor to human and animal health. Recent studies suggest that the immunotoxicity of low dose Cd is not directly apparent, but disrupts the immune responses when infected with some bacteria or virus. But how Cd alters the adaptive immunity organ and cells remains unclear. In this study, we applied lipopolysaccharide (LPS, infectious stressor) to induced inflammation in spleen tissues and T cells, and investigated the effects after Cd exposure and the underlying mechanism. Cd exposure promoted LPS-induced the expressions of the inflammatory factors, induced abnormal initiation of autophagy, but blocked autophagic flux. The effects Cd exposure under LPS activation were reversed by the autophagy promoter Rapamycin. Under LPS activation conditions, Cd also induced oxidative stress by increasing the levels of reactive oxygen species (ROS) and malondialdehyde (MDA), and reducing total antioxidant capacity (T-AOC) activity. The increased superoxide dismutase (SOD) activity after Cd exposure might be a negative feedback or passive adaptive regulation of oxidative stress. Cd-increased autophagic flux inhibition and TNF-α expression were reversed by ROS scavenger α-tocopherol (TCP). Furthermore, under LPS activation condition, Cd promoted activation of toll-like receptor 4 (TLR4)/IκBα/NFκ-B signaling pathway and increased TLR4 protein stability, which were abolished by the pretreatment of Rapamycin. The present study confirmed that, by increasing ROS-mediated inhibiting autophagic degradation of TLR4, Cd promoted LPS-induced inflammation in spleen T cells. This study identified the mechanism of autophagy in Cd-aggravated immunotoxicity under infectious stress, which could arouse public attention to synergistic toxicity of Cd and bacterial or virus infection.

Comparative analysis of immunological changes following realgar and arsenic trioxide treatments in a murine model of myelodysplastic syndrome.

BACKGROUND: Myelodysplastic syndrome (MDS) is a prevalent hematological neoplastic disorder in clinics and its immunopathogenesis has garnered growing interest. Oral and intravenous arsenic agents have long been used to treat hematological malignancies. The main component of oral arsenic is realgar (arsenic disulfide), while arsenic trioxide is the main component of intravenous arsenic. METHODS: This study aimed to assess the effects of ATO and Realgar on the enhancement of peripheral blood, drug safety, and T cell immune status in the NUP98-HOXD13 (NHD13) mice model of MDS, specifically in the peripheral blood, spleen, and liver. RESULTS: The study findings indicate that realgar and arsenic trioxide (ATO) can improve peripheral hemogram in mice, whereas realgar promotes higher peripheral blood cell production than ATO. Furthermore, the clinical administration method and dose did not cause significant toxicity or side effects and thus can be considered safe. Coexistence and interconversion of hyperimmune function and immunosuppression in mice were also observed in this study. In addition, there were interactions between immune cells in the peripheral blood, spleen, and liver to regulate the immune balance of the body and activate immunity via T-cell activation. CONCLUSION: In summary, oral and intravenous arsenic agents are beneficial in improving peripheral hemogram and immunity in mice.

Immunotoxicity of ionic liquid [C14mim]BF4 in rats.

Ionic liquid tetrafluoroborated-1-tetradecyl-3-methylimidazole salt ([C14mim]BF4) immunotoxicity was investigated in rats using three exposure groups (12.5, 25, and 50 mg kg-1), one recovery group (50 mg kg-1), and a control group without any treatment. The findings demonstrated that, at low doses, [C14mim]BF4 could raise WBC, NEU, and MID and lysozyme levels as well as spleen T-lymphocyte stimulation index in rats, however at high doses, the aforementioned indices were dramatically lowered. As the dose was raised, the proportion of RBC and PLT in the blood as well as CD4+ and CD8+ in the spleen increased, but the quantity of immunoglobulin IgG, IgA, and IgM in the serum as well as the number of NK cells in the spleen considerably dropped. Even though there were varying degrees of improvement 30 days after ceasing exposure, all these changes were unable to return to normal, and the number of NK cells was further decreased. The study demonstrates that [C14mim]BF4 can damage the specific immunity and non-specific immunity of rats, and cause immune dysfunction.

The Impact of the Combined Effect of Inhalation Anesthetics and Iron Dextran on Rats' Systemic Toxicity.

Disruption of any stage of iron homeostasis, including uptake, utilization, efflux, and storage, can cause progressive damage to peripheral organs. The health hazards associated with occupational exposure to inhalation anesthetics (IA) in combination with chronic iron overload are not well documented. This study aimed to investigate changes in the concentration of essential metals in the peripheral organs of rats after iron overload in combination with IA. The aim was also to determine how iron overload in combination with IA affects tissue metal homeostasis, hepcidin-ferritin levels, and MMP levels according to physiological, functional, and tissue features. According to the obtained results, iron accumulation was most pronounced in the liver (19×), spleen (6.7×), lungs (3.1×), and kidneys (2.5×) compared to control. Iron accumulation is associated with elevated heavy metal levels and impaired essential metal concentrations due to oxidative stress (OS). Notably, the use of IA increases the iron overload toxicity, especially after Isoflurane exposure. The results show that the regulation of iron homeostasis is based on the interaction of hepcidin, ferritin, and other proteins regulated by inflammation, OS, free iron levels, erythropoiesis, and hypoxia. Long-term exposure to IA and iron leads to the development of numerous adaptation mechanisms in response to toxicity, OS, and inflammation. These adaptive mechanisms of iron regulation lead to the inhibition of MMP activity and reduction of oxidative stress, protecting the organism from possible damage.

Splenic Elemental Composition of Breast Cancer-Suffering Rats Supplemented with Pomegranate Seed Oil and Bitter Melon Extract.

The aim of this study was to investigate how dietary modifications with pomegranate seed oil (PSO) and bitter melon aqueous extract (BME) affect mineral content in the spleen of rats both under normal physiological conditions and with coexisting mammary tumorigenesis. The diet of Sprague-Dawley female rats was supplemented either with PSO or with BME, or with a combination for 21 weeks. A chemical carcinogen (7,12-dimethylbenz[a]anthracene) was applied intragastrically to induce mammary tumors. In the spleen of rats, the selected elements were determined with a quadrupole mass spectrometer with inductively coupled plasma ionization (ICP-MS). ANOVA was used to evaluate differences in elemental composition among experimental groups. Multivariate statistical methods were used to discover whether some subtle dependencies exist between experimental factors and thus influence the element content. Experimental factors affected the splenic levels of macroelements, except for potassium. Both diet modification and the cancerogenic process resulted in significant changes in the content of Fe, Se, Co, Cr, Ni, Al, Sr, Pb, Cd, B, and Tl in rat spleen. Chemometric analysis revealed the greatest impact of the ongoing carcinogenic process on the mineral composition of the spleen. The obtained results may contribute to a better understanding of peripheral immune organ functioning, especially during the neoplastic process, and thus may help develop anticancer prevention and treatment strategies.

Immunological, histological and immunohistochemical alternations induced by zinc oxide nanoparticles and mureer plant in spleen albino rats with the prospective anti-inflammatory action of gallic acid.

The current study was proposed to evaluate the mortal impacts of either alone or mixed treatments of zinc oxide nanoparticles (ZnO NPs) and mureer or Senecio glaucus L. plant (SP) on spleen tissue via immunological and histological studies and to estimate the likely immunomodulatory effect of gallic acid (GA) for 30 days in rats. Rats were classified into eight groups with orally treated: Control, GA (100mg/kg), ZnO NPs (150mg/kg), SP (400mg/kg), GA+ZnO NPs (100,150mg/kg), GA+SP (100,400mg/kg), ZnONPs+SP (150,400mg/kg) and GA+ZnONPs+SP (100,150,400mg/kg). Interleukin-6 (IL-6) level was measured using an enzyme-linked immunoassay (ELISA). Also, the pro-apoptotic protein (caspase-3) expression was estimated using an immunohistochemistry assay. Our data revealed that ZnO NPs and SP triggered a significant increase in the levels of IL-6 and total lipids (TL) and the activity of lactate dehydrogenase (LDH), (p<0.001). Furthermore, they overexpressed caspase-3 and caused lymphoid depletion. They revealed that the immunotoxic outcome of mixed treatment was more than the outcome of the alone treatment. However, GA restored the spleen damage from these adverse results. Finally, this study indicated that ZnO NPs and SP might be immunotoxic and splenotoxic agents; however, GA may be displayed as an anti-inflammatory and splenic-protective agent.

Xuanhusuo powder has an anti-breast cancer effect by inhibiting myeloid-derived suppressor cell differentiation in the spleen of mice through down-regulating granulocyte colony stimulating factor. / çŽ„èƒ¡ç´¢æ•£é€šè¿‡ä¸‹è°ƒç²’ç»†èƒžé›†è½åˆºæ¿€å› å­æŠ‘åˆ¶è„¾è„é«“æºæŠ‘åˆ¶ç»†èƒžåˆ†åŒ–å‘æŒ¥æŠ—ä¹³è ºç™Œä½œç”¨.

OBJECTIVES: To investigate the mechanism of Xuanhusuo powder (XHSP) inhibiting the differentiation of spleen myeloid-derived suppressor cells (MDSCs) in breast cancer mice. METHODS: Forty-eight BALB/c female mice aged 4-5 weeks were selected, 6 of them were in normal control group, while others were in tumor-bearing models established by orthotopic injection of 4T1 cells into the subcutaneous fat pad of the second pair of left mammary glands. The tumor-bearing mice were divided into granulocyte colony stimulating factor (G-CSF) control group, G-CSF knock-down group, model control group, XHSP small dose group, XHSP medium dose group, XHSP high dose group, and cyclophosphamide (CTX) group, with 6 mice in each group. G-CSF control group and G-CSF knock-down group were constructed by stably transfecting 4T1 cells established by shRNA lentivirus combined with puromycin selection. 48 h after the model was established, XHSP small, medium, high dose group were given 2, 4, 8 g·kg－1·d－1 intragastric administration once a day, respectively. CTX was given 30 mg/kg by intraperitoneal injection, once every other day. The other groups were given an equal volume of 0.5% hydroxymethylcellulose sodium. The drugs in each group were continuously administered for 25 d. Histological changes in spleen were observed by HE staining, the proportion of MDSCs subsets in the spleen were detected by flow cytometry, the co-expression of CD11b and Ly6G in the spleen was detected by immunofluorescence, and the concentration of G-CSF in peripheral blood was detected by ELISA. The spleen of tumor-bearing mice was co-cultured with 4T1 stably transfected cell lines in vitro, treated with XHSP (30 µg/mL) for 24 h, and the co-expression of CD11b and Ly6G in the spleen was detected by immunofluorescence. 4T1 cells were treated by XHSP (10, 30, 100 µg/mL) for 12 h. The mRNA level of G-CSF was detected by realtime RT-PCR. RESULTS: Compared with normal mice, the red pulp of the spleen in tumor-bearing mice was widened with megakaryocyte infiltration. The proportion of spleen polymorphonucleocyte-like MDSCs (PMN-MDSCs) was significantly increased (P<0.01) and the co-expression of CD11b and Ly6G was increased, and the concentration of G-CSF in peripheral blood was significantly increased (P<0.01). However, XHSP could significantly reduce the proportion of PMN-MDSCs (P<0.05) and the co-expression of CD11b and Ly6G in the spleen, down-regulate the mRNA level of G-CSF in 4T1 cells (P<0.01). The concentration of G-CSF in peripheral blood of tumor-bearing mice also decreased (P<0.05) and tumor volume was reduced and splenomegaly was improved (all P<0.05). CONCLUSIONS: XHSP may play an anti-breast cancer role by down-regulating G-CSF, negatively regulating the differentiation of MDSCs, and reconstruct the spleen myeloid microenvironment.

Soluble thrombomodulin ameliorates aberrant hemostasis after rewarming in a rat accidental hypothermia model.

BACKGROUND: Accidental hypothermia (AH) sometimes leads to coagulation disorder, especially in severe AH. We previously demonstrated that intrasplenic platelet activation caused aberrant hemostasis and thrombus formation after rewarming in a murine AH model. However, no study has focused on the appropriate management of platelets causing coagulation activation after rewarming of AH. We investigated whether or not recombinant soluble thrombomodulin (rTM) can suppress thrombosis formation after rewarming using a rat AH model. METHODS: Wistar rats were exposed to an ambient temperature of -20 °C under general anesthesia until their rectal temperature decreased to 26 °C. The Hypo group rats (n = 5) were immediately euthanized, while the Hypo/Re group (n = 5) and rTM group rats (n = 5), which were administered rTM (1 mg/kg) via the tail vein, were rewarmed until the rectal temperature returned to 34 °C and then euthanized 6 h later. Tissue and blood samples were collected from all rats for histopathological and coagulation analyses at euthanasia. RESULTS: There was no significant change in the D-dimer level in the Hypo group rats, while the D-dimer level was significantly elevated at 6 h after rewarming in the Hypo/Re group rats (P = 0.015), and histopathology detected both fibrin and platelets in the renal glomerulus. However, the rTM group rats did not show any elevation of the D-dimer levels at 6 h after rewarming, and no fibrin was noted on histopathology. CONCLUSIONS: rTM may be useful as an appropriate anticoagulant in cases of aberrant hemostasis after rewarming of AH.

Natural killer cells expanded in vivo or ex vivo with IL-15 overcomes the inherent susceptibility of CAST mice to lethal infection with orthopoxviruses.

The wild-derived inbred CAST/EiJ mouse, one of eight founder strains in the Collaborative Cross panel, is an exceptional model for studying monkeypox virus (MPXV), an emerging human pathogen, and other orthopoxviruses including vaccinia virus (VACV). Previous studies suggested that the extreme susceptibility of the CAST mouse to orthopoxviruses is due to an insufficient innate immune response. Here, we focused on the low number of natural killer (NK) cells in the naïve CAST mouse as a contributing factor to this condition. Administration of IL-15 to CAST mice transiently increased NK and CD8+ T cells that could express IFN-γ, indicating that the progenitor cells were capable of responding to cytokines. However, the number of NK cells rapidly declined indicating a defect in their homeostasis. Furthermore, IL-15-treated mice were protected from an otherwise lethal challenge with VACV or MPXV. IL-15 decreased virus spread and delayed death even when CD4+/CD8+ T cells were depleted with antibody, supporting an early protective role of the expanded NK cells. Purified splenic NK cells from CAST mice proliferated in vitro in response to IL-15 and could be activated with IL-12/IL-18 to secrete interferon-γ. Passive transfer of non-activated or activated CAST NK cells reduced VACV spread but only the latter completely prevented death at the virus dose used. Moreover, antibodies to interferon-γ abrogated the protection by activated NK cells. Thus, the inherent susceptibility of CAST mice to orthopoxviruses can be explained by a low level of NK cells and this vulnerability can be overcome either by expanding their NK cells in vivo with IL-15 or by passive transfer of purified NK cells that were expanded and activated in vitro.

Investigation of the supportive therapy potential of propolis extract and Lactobacillus acidophilus LA-5 milk combination against breast cancer in mice.

Immunotherapy has been applied in cancer treatments for many years as an alternative treatment method to radiotherapy, chemotherapy. It is well known that immunotherapy could suppress tumor formation by modulating the immune system of the host. The aim of the study is to investigate supportive therapy potential of acidophilus milk (AS) and propolis extract (PE) in the mouse xenograft breast cancer model. For this purpose, firstly cytotoxic effect of PE was determined by MTT assay against 4 T1 mouse breast cancer cells. Apoptotic effect of PE analyzed by flow cytometry. The antibacterial activity of PE was determined by the 96-well microplate broth-dilution method on Lactobacillus acidophilus LA-5. Then, Balb/c mice were injected subcutaneously with 4 T1 cells (2x105 cells/mouse) and also mice were given daily oral gavage with PE (66 mg/kg/day) and/or acidophilus milk (108 CFU/mL/mouse/day) for 14 days. The Balb/c mice were weighed throughout the study, and the tumor sizes were measured by caliper at the 14th day. The proliferation of splenocytes which collected spleen from mice was measured by MTT. CD8 + T cell response was analyzed by flow cytometry and results were evaluated in comparison with control and tumor control groups. The IC50 value for PE on 4 T1 cells was determined as 129.25 ± 1.90 µg/mL. The apoptotic effect of PE at IC50 concentration was determined as 3.3% of cells to late-apoptosis, 4.3% of cells to pro-apoptosis and 2.5% of cells to necrosis. The MIC and MBC values for PE on L. acidophilus LA-5 were 5000 ppm. The treatment of PE, AS and the combination of PE and AS were inhibited the tumor volumes by 59.16%, 28.29% and 63.39%, respectively. Acidophilus milk and PE combination significantly enhanced the ConA-, LPS- and PHA-induced splenocyte proliferation (P < 0.05). The acidophilus milk and PE combination were also found to stimulate IFN- γ production. In conclusion, the best anti-tumor effect was obtained by the combination of acidophilus milk and propolis.

Traditional Patchouli essential oil modulates the host's immune responses and gut microbiota and exhibits potent anti-cancer effects in ApcMin /+ mice.

Patchouli Essential Oil (PEO) has been used as a scent for various healing purposes since the ancient Egyptian period. The primary source of the oil is Pogostemon cablin (PC), a medicinal plant for treating gastrointestinal symptoms. However, the pharmacological function has not been addressed. Here, we report the cancer prevention and gut microbiota (GM) modulating property of PEO and its derivatives patchouli alcohol (PA) and pogostone (PO) in the ApcMin /+ colorectal cancer mice model. We found that PEO, PA, and PO significantly reduced the tumor burden. At the same time, it strengthened the epithelial barrier, evidenced by substantially increasing the number of the goblet and Paneth cells and upregulation of tight junction and adhesion molecules. In addition, PEO, PA, and PO shifted M1 to M2 macrophage phenotypes and remodeled the inflammatory milieu of ApcMin /+ mice. We also found suppression of CD4+CD25+ and stimulation CD4+ CD8+ cells in the spleen, blood, mesenteric lymph nodes (MLNs), and Peyer's patches (PPs) of the treated mice. The composition of the gut microbiome of the drug-treated mice was distinct from the control mice. The drugs stimulated the short-chain fatty acids (SCFAs)-producers and the key SCFA-sensing receptors (GPR41, GPR43, and GPR109a). The activation of SCFAs/GPSs also triggered the alterations of PPAR-γ, PYY, and HSDCs signaling mediators in the treated mice. Our work showed that PEO and its derivatives exert potent anti-cancer effects by modulating gut microbiota and improving the intestinal microenvironment of the ApcMmin /+ mice.

The Bile Acid Receptor GPBAR1 Modulates CCL2/CCR2 Signaling at the Liver Sinusoidal/Macrophage Interface and Reverses Acetaminophen-Induced Liver Toxicity.

Drug-induced liver injury caused by acetaminophen (acetyl-para-aminophenol [APAP]) is the main cause of acute liver failure and liver transplantation in several Western countries. Whereas direct toxicity exerted by APAP metabolites is a key determinant for early hepatocytes injury, the recruitment of cells of innate immunity exerts a mechanistic role in disease progression, determining the clinical outcomes. GPBAR1 is a G protein-coupled receptor for secondary bile acids placed at the interface between liver sinusoidal cells and innate immunity. In this report, using genetic and pharmacological approaches, we demonstrate that whereas Gpbar1 gene deletion worsens the severity of liver injury, its pharmacological activation by 6ß-ethyl-3a,7b-dihydroxy-5b-cholan-24-ol rescues mice from liver injury caused by APAP. This protective effect was supported by a robust attenuation of liver recruitment of monocyte-derived macrophages and their repolarization toward an anti-inflammatory phenotype. Macrophage depletion by gadolinium chloride pretreatment abrogated disease development, whereas their reconstitution by spleen-derived macrophage transplantation restored the sensitivity to APAP in a GPBAR1-dependent manner. RNA sequencing analyses demonstrated that GPBAR1 agonism modulated the expression of multiple pathways, including the chemokine CCL2 and its receptor, CCR2. Treating wild-type mice with an anti-CCL2 mAb attenuated the severity of liver injury. We demonstrated that negative regulation of CCL2 production by GPBAR1 agonism was promoter dependent and involved FOXO1. In conclusion, we have shown that GPBAR1 is an upstream modulator of CCL2/CCR2 axis at the sinusoidal cell/macrophage interface, providing a novel target in the treatment of liver damage caused by APAP.

Activation of CB1R Promotes Lipopolysaccharide-Induced IL-10 Secretion by Monocytic Myeloid-Derived Suppressive Cells and Reduces Acute Inflammation and Organ Injury.

Cannabis sativa and its principal components, Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol, are increasingly being used to treat a variety of medical problems, including inflammatory conditions. Although studies suggest that the endocannabinoid system has immunomodulatory properties, there remains a paucity of information on the effects of cannabinoids on immunity and on outcomes of infection and injury. We investigated the effects and mechanism(s) of action of cannabinoid receptor agonists, including Δ9-THC, on inflammation and organ injury in endotoxemic mice. Administration of Δ9-THC caused a dramatic early upregulation of plasma IL-10 levels, reduced plasma IL-6 and CCL-2 levels, led to better clinical status, and attenuated organ injury in endotoxemic mice. The anti-inflammatory effects of Δ9-THC in endotoxemic mice were reversed by a cannabinoid receptor type 1 (CB1R) inverse agonist (SR141716), and by clodronate-induced myeloid-cell depletion, but not by genetic invalidation or blockade of other putative Δ9-THC receptors, including cannabinoid receptor type 2, TRPV1, GPR18, GPR55, and GPR119. Although Δ9-THC administration reduced the activation of several spleen immune cell subsets, the anti-inflammatory effects of Δ9-THC were preserved in splenectomized endotoxemic mice. Finally, using IL-10-GFP reporter mice, we showed that blood monocytic myeloid-derived suppressive cells mediate the Δ9-THC-induced early rise in circulating IL-10. These results indicate that Δ9-THC potently induces IL-10, while reducing proinflammatory cytokines, chemokines, and related organ injury in endotoxemic mice via the activation of CB1R. These data have implications for acute and chronic conditions that are driven by dysregulated inflammation, such as sepsis, and raise the possibility that CB1R-signaling may constitute a novel target for inflammatory disorders.