Splenic IL-6 Research Articles

No-observed adverse effect levels of deoxynivalenol and aflatoxin B1 in combination induced immune inhibition and apoptosis in vivo and in vitro

Mycotoxins are toxic metabolites produced by fungal species, commonly exist in animal feeds, and pose a serious risk to human as well as animal health. But limited studies have focused on combined effects of no-observed adverse effect levels. In vivo study, 6 weeks old twenty-four mice were individually exposed to Deoxynivalenol (DON) at 0.1 mg/kg BW, Aflatoxin B1 (AFB1) at 0.01 mg/kg BW, and mixture of DON and AFB1 (0.1 mg/kg BW and 0.01 mg/kg BW, respectively) for 28 days. Then, DON at 0.5 μg/mL, AFB1 at 0.04 μg/mL, and mixtures of DON and AFB1 (0.5 μg/mL, 0.04 μg/mL, respectively) were applied to porcine alveolar macrophages (PAMs) in vitro study. Our in vivo results revealed that the combined no-observed adverse effect levels of DON and AFB1 administration decreased IgA and IgG levels in the serum, the splenic TNF-α, IFN-γ, IL-2 and IL-6 mRNA expression and T-lymphocyte subset levels (CD4+ and CD8+) in the spleen. Additionally, the combined administration increased caspase-3, caspase-9, Bax, Cyt-c, and decreased Bcl-2 protein expression. Taken together, the combined no-observed adverse effect levels of DON and AFB1 could induce immunosuppression, which may be related to apoptosis. This study provides new insights into the combined immune toxicity (DON and AFB1).

The mechanism of Xihuang pills' intervention in the tumour immune microenvironment for the treatment of liver cancer based on the STAT3-PDL1 pathway

Ethnopharmacological relevanceXihuang pills, a time-honored Chinese compound formula with a history spanning thousands of years, have demonstrated remarkable efficacy in treating various cancers, such as breast cancer, colon cancer, and liver cancer. Clinical applications over the years have established their effectiveness. Several scholars conducting experimental studies have elucidated the potent tumor-suppressing effects of Xihuang pills. While the inhibition of tumor vascular development and prevention of tumor cell invasion and metastasis have been well-explored mechanisms, the impact on the tumor immune microenvironment has received less attention. This study focuses on investigating the immune microenvironment adjustments induced by Xihuang pills in hepatocellular carcinoma. Aim of the studyTumour cells will find an escape phenomenon during tumour immunotherapy, which will affect immunotherapy results. We will research the regulation of the tumour immune microenvironment, to provide a more complete and precise basis for the elucidation of the mechanism of Xihuang pills in treating cancers. It provides new research ideas for people to treat liver cancer. Materials and methodsThrough in vivo and in vitro assessments confirming the intervention effects of Xihuang pills, we observed alterations in T cell typing, macrophage polarization, and tumor-associated cytokine levels. The primary active ingredients of Xihuang pills were identified using UPLC-MS/GC-MS, and relevant pathways in the treatment of hepatocellular carcinoma were predicted through network pharmacology. Combining the network pharmacology approach, we predicted the pathways relevant to Xihuang pills in treating hepatocellular carcinoma and experimentally validated the involvement of PD-1/PD-L1, a key immunity-related axis. ResultsXihuang Pill has a regulatory effect on the tumor immune microenvironment. ConclusionsThe results indicated that Xihuang pills could impact splenic lymphocyte phenotyping, macrophage polarization, and IL-6 cytokine expression in liver cancer mice. The mechanism of action was associated with the regulation of the PD-1/PD-L1 signaling pathway by the STAT3 protein.

Low Intensity Renal Ultrasound Exposure Reduces Systemic Inflammation Induced by LPS Challenge in Mice

Low-intensity pulsed ultrasound (LIPUS), a procedure historically used to stimulate bone healing and tissue repair, activates the splenic cholinergic anti-inflammatory pathway and ultimately reduces circulating and tissue-specific pro-inflammatory cytokine release if applied to the kidney prior to renal ischemia/reperfusion injury. We questioned whether LIPUS would be effective in reducing the inflammatory response to lipopolysaccharide (LPS) if exposure occurred as pre- or post-treatment. We hypothesized that LIPUS exposure before an LPS challenge would suppress the subsequent inflammatory response in mice, and that LIPUS exposure after an LPS challenge would be similarly beneficial. Female C57BL/6J mice were randomized into Saline, LPS, LPS/Pre-treatment and LPS/Post-treatment groups at 15 weeks of age. Pre-treated mice were subjected to 20-minute LIPUS exposure while under anesthesia for five consecutive days. On the 6th day, mice were injected with LPS (1 mg/kg dissolved in saline, IP) or saline (100 μL). One hour after LPS injection, post-treated mice were exposed to LIPUS for 20 min. Three hours after LPS or saline injection, all mice were humanely euthanized, and plasma, spleen and kidney were harvested for analysis of proinflammatory cytokines associated with the response to LPS. LPS significantly increased plasma TNF-α (280±37 vs. 0.07±0.00 pg/mL), plasma IL-6 (96124±6688 vs. 425±181 pg/mL), splenic IL-6 (0.77±0.16 vs. 0.17±0.04 pg/μg of protein), and renal cortical IL-6 (0.50±0.07 vs. 0.01±0.00 pg/μg of protein) compared to saline-treated mice (all p<0.05). LIPUS pre-treatment significantly reduced plasma TNF-α (183±15 pg/mL) and plasma IL-6 (63656±8441 pg/mL), without altering splenic and renal cytokines in LPS-treated mice. LIPUS post-treatment lowered plasma IL-6 (47432 ± 1182 pg/mL). In conclusion, five days of LIPUS exposure prior to an LPS challenge effectively reduced circulating inflammatory mediators, while a single LIPUS exposure after an LPS challenge also successfully reduced systemic IL-6. These findings demonstrate the potential in using LIPUS as a therapeutic strategy for quelling acute inflammatory responses and warrant further investigation of its use as a therapy for chronic inflammatory diseases. Studies funded by Department of Defense (LR210096) for KWM. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Regulation of Inflammation, Without Impacting Heart Rate, via Electrical Stimulation of the Dorsal Motor Nucleus of the Vagus

The vagus nerve plays an important role in neuroimmune interactions and in the regulation of inflammation. Recently, using optogenetics it has been demonstrated that the brainstem’s dorsal motor nucleus of the vagus (DMN) is a significant source of efferent vagus nerve fibers that control inflammation. In contrast to optogenetics, electrical neuronal stimulation is an approved therapeutic approach. However, the anti-inflammatory effectiveness of electrical stimulation of the DMN (eDMNS) and the possible heart rate (HR) alterations associated with this approach have not been investigated. Here, we examined the effects of eDMNS on the HR and cytokine levels in murine endotoxemia as well as the cecal ligation and puncture (CLP) model of sepsis. C57BL/6 mice (8-10 weeks old), under anesthesia and secured in a stereotaxic frame, received eDMNS via a concentric bipolar electrode in the left or right DMN, or sham treatment. eDMNS (50, 250 or 500 μA and 30 Hz, for 1 min) was performed and HR recorded. In endotoxemia experiments, mice underwent sham or 5-minute eDMNS (250 μA or 50 μA) followed by LPS (0.5 mg/kg) i.p. injection. eDMNS was also studied on mice with cervical unilateral vagotomy or sham surgery. In CLP experiments sham or left eDMNS was performed immediately post CLP. Cytokines and corticosterone were measured 90 mins after LPS administration or 24h after CLP. CLP survival was monitored for 14 days. Both left and right eDMNS at 250 μA and 500 μA significantly reduced HR, compared with pre- and post-stimulation, while 50 μA did not. Left-side eDMNS at 50 μA decreased serum and splenic TNF levels, and increased serum IL-10 during endotoxemia. The anti-inflammatory effect of eDMNS was abrogated in mice with ipsilateral unilateral vagotomy, which was also not associated with serum corticosterone alterations. Right-side eDMNS lowered serum TNF levels and increased IL-10 but had no impact on splenic cytokines. In CLP, left eDMNS decreased serum TNF and IL-6 levels, lowered splenic IL-6 and increased splenic IL-10, and improved survival in mice. For the first time, we demonstrate that an eDMNS regimen, which does not cause bradycardia, mitigates LPS-induced inflammation. These effects are dependent on an intact vagus nerve and are unrelated to corticosteroid alterations. Additionally, eDMNS reduces inflammation and enhances survival in a polymicrobial sepsis model. These findings hold promise for further research into bioelectronic anti-inflammatory strategies aimed at the brainstem DMN. This work was partially funded by NIGMS. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Electrical stimulation of the dorsal motor nucleus of the vagus in male mice can regulate inflammation without affecting the heart rate

BackgroundThe vagus nerve plays an important role in neuroimmune interactions and in the regulation of inflammation. A major source of efferent vagus nerve fibers that contribute to the regulation of inflammation is the brainstem dorsal motor nucleus of the vagus (DMN), as recently shown using optogenetics. In contrast to optogenetics, electrical neuromodulation has broad therapeutic implications. However, the anti-inflammatory effectiveness of electrical stimulation of the DMN (eDMNS) and the possible heart rate (HR) alterations associated with this approach have not been investigated. Here, we examined the effects of eDMNS on HR and cytokine levels in mice administered with lipopolysaccharide (LPS, endotoxin) and in mice subjected to cecal ligation and puncture (CLP) sepsis. MethodsAnesthetized male 8–10-week-old C57BL/6 mice on a stereotaxic frame were subjected to eDMNS using a concentric bipolar electrode inserted into the left or right DMN or sham stimulation. eDMNS (500, 250 or 50 μA at 30 Hz, for 1 min) was performed and HR recorded. In endotoxemia experiments, sham or eDMNS utilizing 250 μA or 50 μA was performed for 5 mins and was followed by LPS (0.5 mg/kg) i.p. administration. eDMNS was also applied in mice with cervical unilateral vagotomy or sham operation. In CLP experiments sham or left eDMNS was performed immediately post CLP. Cytokines and corticosterone were analyzed 90 mins after LPS administration or 24 h after CLP. CLP survival was monitored for 14 days. ResultsEither left or right eDMNS at 500 μA and 250 μA decreased HR, compared with baseline pre-stimulation. This effect was not observed at 50 μA. Left side eDMNS at 50 μA, compared with sham stimulation, significantly decreased serum and splenic levels of the pro-inflammatory cytokine TNF and increased serum levels of the anti-inflammatory cytokine IL-10 during endotoxemia. The anti-inflammatory effect of eDMNS was abrogated in mice with unilateral vagotomy and was not associated with serum corticosterone alterations. Right side eDMNS in endotoxemic mice suppressed serum TNF and increased serum IL-10 levels but had no effects on splenic cytokines. In mice with CLP, left side eDMNS suppressed serum IL-6, as well as splenic IL-6 and increased splenic IL-10 and significantly improved the survival rate of CLP mice. ConclusionsFor the first time we show that a regimen of eDMNS which does not cause bradycardia alleviates LPS-induced inflammation. These eDMNS anti-inflammatory effects require an intact vagus nerve and are not associated with corticosteroid alterations. eDMNS also decreases inflammation and improves survival in a model of polymicrobial sepsis. These findings are of interest for further studies exploring bioelectronic anti-inflammatory approaches targeting the brainstem DMN.

IL6 suppresses vaccine responses in neonates by enhancing IL2 activity on T follicular helper cells

The inability of neonates to develop CD4+FoxP3-CXCR5hiPD-1hi T follicular helper (TFH) cells contributes to their weak vaccine responses. In previous studies, we measured diminished IgG responses when IL-6 was co-injected with a pneumococcal conjugate vaccine (PCV) in neonatal mice. This is in sharp contrast to adults, where IL-6 improves vaccine responses by downregulating the expression of IL-2Rβ on TFH cells and protecting them from the inhibitory effect of IL-2. In this study, we found that splenic IL-6 levels rapidly increased in both adult and neonatal mice following immunization, but the increase in neonatal mice was significantly more than that of adult mice. Moreover, immunized neonatal TFH cells expressed significantly more IL-2 as well as its receptors, IL-2Rα and IL-2Rβ, than the adult cells. Remarkably, IL-6 co-injection with PCV vaccine further increased the production of IL-2 and the expression of its receptors by neonatal TFH cells, whereas excess IL-6 had totally opposite effect in immunized adult mice. Underscoring the role of IL-6 in activating the IL-2 mediated suppression of vaccine responses, immunization of IL-6 knock-out neonates led to improved antibody responses accompanied by expanded TFH cells as well as lower levels of IL-2 and IL-2 receptors on TFH cells. Moreover, CpG containing PCV improved TFH response in neonates by suppressing the expression of IL-2 receptors on TFH cells and inhibiting IL-2 activity. These findings unveil age-specific differences in IL-6 mediated vaccine responses and highlight the need to consider age-related immunobiological attributes in designing vaccines.

An atopic dermatitis-like murine model by skin-brushed cockroach Per a 2 and oral tolerance induction by Lactococcus lactis-derived Per a 2.

Atopic dermatitis (AD) is a complex, chronic inflammatory skin disease. An estimated 57.5% of asthmatic patients and 50.7% of rhinitis patients are allergic to cockroaches in Taiwan. However, the role of cockroaches in the pathogenesis of AD is undetermined. Oral tolerance might be another strategy for protecting against AD and allergic inflammation by regulating T helper 2 (Th2) immune responses. Aim to examine the underlying immunologic mechanism, we developed an AD-like murine model by skin-brushing with cockroach Per a 2. We also investigated whether the systemic inflammation of AD in this murine model could be improved by specific tolerance to Lactococcus lactis-expressing Per a 2, which was administered orally. Repeated painting of Per a 2 without adjuvant to the skin of mice resulted in increased total IgE, Per a 2-specific IgE, and IgG1, but not IgG2a. In addition, epidermal thickening was significantly increased, there were more scratch episodes, and there were increases in total white blood cells (eosinophil, neutrophil, and lymphocyte) and Th2 cytokines (Interleukin (IL)-4, IL-5, IL-9, and IL-13) in a dose-dependent manner. The results revealed that oral administration of L. lactis-Per a 2 ameliorated Per a 2-induced scratch behavior and decreased the production of total IgE, Per a 2-specific IgE, and IgG1. Furthermore, L. lactis-Per a 2 treatment also suppressed inflammatory infiltration, expressions of thymic stromal lymphopoietin (TSLP) and IL-31 in skin lesions, and downregulated splenic IL-4 and IL-13 in Per a 2-induced AD mice. This study provides evidence supporting that repeated brushing of aeroallergens to the skin leads to atopic dermatitis phenotypes and oral allergen-specific immune tolerance can ameliorate AD-like symptoms and systemic inflammation and prevent progression of atopic march.

Abstract P3158: Extracellular Vesicles From Ischemic Myocardium Mediate Regulatory T Cell Dysfunction Through MicroRNA-1-mediated Suppression Of VEGF-A

Motivation: Myocardial infarction (MI) triggers an efflux of pro-inflammatory extracellular vesicles (EVs) from the heart into the circulation. Here, we test the hypothesis that circulating ischemic heart EVs (iEVs) attenuate regulatory T cell (Treg) proliferation and function post-MI. Methods: Hearts were explanted from mice and Langendorff-perfused with Tyrode buffer either continuously for two hours (nonischemic), or subjected to no-flow for 30 min, and reperfused for 1.5h. Heart effluent (~700ml) was collected for EV isolation by ultracentrifugation. EV size and concentration were measured by nanoparticle tracking analysis; EV markers were characterized by western blot. EVs from nonischemic heart effluent (nEVs) served as controls. RNA sequencing (RNAseq) followed by qPCR validation was used to identify microRNAs in EVs. Assessments of human Tregs included cell proliferation (Cell Counting Kit-8 assay), IL-10 production (qPCR of Tregs, and ELISA of Treg-conditioned media), and RNAseq after exposure to EVs. In vivo, an antagomir was administered retro-orbitally immediately post-MI, and splenic Tregs were measured by flow cytometry 7 days post-MI. Findings: MiR-1-3p was highly enriched in iEVs compared to nEVs. Exposure of human Tregs to iEVs or a miR-1 mimic resulted in decreased proliferation ( P <0.0001, N=8) and production of IL-10 (qPCR: P<0.0001, n=9; ELISA: P <0.0001, N=6) compared to nEVs. Antagomir-1 reversed iEV- and miR-1-induced inhibition of Treg proliferation. RNAseq on human Tregs revealed that VEGF-A, a miR-1 target gene, was suppressed after exposure to iEVs compared to nEVs. In vivo, the number of splenic Tregs decreased on day 7 post-MI, with a concomitant decrease in IL-10 + and VEGF-A + Tregs. Intriguingly, administration of antagomiR-1 restored numbers of splenic IL10 + and VEGF-A + Treg cells. Conclusions: iEVs cause Treg dysfunction by transferring miR-1. In turn, miR-1 suppresses VEGF-A, a known modulator of Treg function. Our findings reveal novel paracrine modulation of immune cells by ischemic myocardium. Targeting EV signaling post-MI represents a potential new strategy to augment Treg-mediated tissue repair.

SPLENIC INVARIANT NATURAL KILLER T CELLS PLAY A SIGNIFICANT ROLE IN THE RESPONSE TO POLYMICROBIAL SEPSIS.

Background: Sepsis is marked by a dysregulated immune response to an infection. Invariant natural killer T cells ( i NKT cells) are a pluripotent lymphocyte subpopulation capable of affecting and coordinating the immune response to sepsis. The spleen is an important site of immune interactions in response to an infection. Splenic i NKT cells have emerged as important potential frontline mediators of chronic immune response. There are few data addressing the role splenic of i NKT cells in response to intra-abdominal polymicrobial sepsis. Methods: The cecal ligation and puncture model was used to create intra-abdominal sepsis in 8- to 12-week-old wild-type, i NKT -/- , or programmed cell death receptor-1 (PD-1) -/- mice. Twenty-four hours later, spleens were harvested. Flow cytometry was used for phenotyping using monoclonal antibodies. Cell sort was used to isolate i NKT cells. A macrophage cell line was used to assess i NKT cell-phagocyte interactions. Enzyme-linked immunosorbent assay was used for cytokine analysis. Results: Splenic i NKT-cell populations rapidly declined following induction of sepsis. Within i NKT-cell -/- mice, a distinct baseline hyperinflammatory environment was noted. Within wild type, sepsis induced an increase in splenic IL-6 and IL-10, whereas in i NKT -/- mice, there was no change in elevated IL-6 levels and a noted decrease in IL-10 expression. Further, following sepsis, PD-1 expression was increased upon spleen i NKT cells. With respect to PD-1 ligands upon phagocytes, PD-1 ligand expression was unaffected, whereas PD-L2 expression was significantly affected by the presence of PD-1. Conclusions: Invariant natural killer T cells play a distinct role in the spleen response to sepsis, an effect mediated by the checkpoint protein PD-1. Given that modulators are available in clinical trials, this offers a potential therapeutic target in the setting of sepsis-induced immune dysfunction.

Effects of dietary nano-selenium supplementation on Riemerella anatipestifer vaccinated and challenged Pekin ducklings (Anas platyrhynchos)

Riemerella anatipestifer (RA) is a common disease causing economic losses to duck farms worldwide. Novel supplements are crucially needed to control this bacterium, enhance poultry performance, and produce synergistic effects with vaccines in stimulating the immune system. This study investigated the effect of nano-selenium (Nano-Se) on the vaccinated (VAC) and challenged (Ch) Pekin ducklings (Anas platyrhynchos) with RA. Five experimental groups (G1-G5) were included in this study: G1 was the control group, G2 was the RA-challenged group, G3 was the Nano-Se+Ch group, G4 was the VAC+Ch group, and G5 was the Nano-Se+VAC+Ch group. The Nano-Se (0.3 mg/kg diet) was supplemented for 5 weeks post-vaccination (PV). The ducklings were vaccinated subcutaneously with the RA vaccine at 7 days of age and challenged with RA at the 3rd week PV. Blood, pharyngeal swabs and tissue samples were collected at the 3rd week PV and at different times post-challenge (PC). The growth performance (weight gain and feed conversion ratio), clinical signs, gross lesions, mortality, bacterial shedding, haematological, immunological, and biochemical parameters, cytokines production, and histopathological lesion scores showed significant differences (P < 0.05) between the challenged (G2) group and the supplemented (G3 & G5) groups. G5 showed the highest (P < 0.05) growth performance, phagocytic activity, IgM and IgG, splenic interleukin-2 (IL-2), IL-10, and interferon-gamma (IFN-γ) gene expressions, and the lowest mortality, bacterial shedding, hepatic and renal damage, heterophil/lymphocyte ratio and lesion scores compared to the other groups. In conclusion, the supplementation of nano-selenium for five weeks in the diet can improve the growth performance, immune status, and cytokines production in ducklings vaccinated and challenged with RA.

IL-6 suppresses vaccine responses in neonatal mice by enhancing IL-2 activity on T follicular helper cells

Abstract The inability of neonates to develop CD4+CXCR5+PD-1+ T follicular helper (TFH) cells contributes to their weak vaccine responses. In previous studies, we measured diminished IgG responses when IL-6 was co-injected with a pneumococcal conjugate vaccine (PCV) in neonatal mice. This is in sharp contrast to adults, where IL-6 improves vaccine responses by downregulating the expression of IL-2Rβ on TFH cells and protecting them from the inhibitory effect of IL-2. In this study, we found that splenic IL-6 levels rapidly increased in both adult and neonatal mice following immunization, but the increase in neonatal mice was significantly more than that of adult mice. Moreover, immunized neonatal TFH cells expressed significantly more IL-2 as well as its receptors, IL-2Rα and IL-2Rβ, than the adult cells. Remarkably, IL-6 co-injection with PCV vaccine further increased the production of IL-2 and the expression of its receptors by neonatal TFH cells, whereas excess IL-6 had totally opposite effect in immunized adult mice. Underscoring the role of IL-6 in activating the IL-2 mediated suppression of vaccine responses, immunization of IL-6 knock-out neonates led to improved antibody responses accompanied by expanded TFH cells as well as lower levels of IL-2 and IL-2 receptors on TFH cells. Moreover, CpG containing PCV improved TFH response in neonates by suppressing the expression of IL-2 receptors on TFH cells and inhibiting IL-2 activity. These findings unveil age-specific differences in IL-6 mediated vaccine responses and highlight the need to consider age-related immunobiological attributes in designing vaccines. This study was funded by US FDA, Perinatal Health Center of Excellence (PHCE) grant to Mustafa Akkoyunlu as well as intramural grants from US FDA.

A58 ENTERIC TUFT CELLS MODULATE LOCAL BUT NOT SYSTEMIC HOST IMMUNE RESPONSES TO CO-ORDINATE TIMELY EXPULSION OF HYMENOLEPIS DIMINUTA

Abstract Background The intestinal tuft cell is a versatile epithelial cell implicated in host detection and defence against enteric nematode and trematode parasites by producing interleukin(IL)-25 and cysteinyl leukotrienes. We have shown that immunocompetent mice develop small intestinal tuft cell hyperplasia during infection with the cestode, Hymenolepis diminuta. Whether tuft cells coordinate murine expulsion of H. diminuta and if this response is similar to other well-established parasitic models is unknown. Purpose To test 1) if tuft cells coordinate host detection and immune responses to H. diminuta; and, 2) if the lack of functional B, T cells and innate lymphoid cells (ILC2s) affect tuft cell hyperplasia and expulsion of the parasite. Method Male and female pou2f3-/-/-/+ littermates, rag-1-/- and C57BL6 mice were infected with 5 cysticercoids of H. diminuta. ILC2s were depleted in rag-1-/- with anti-CD90.2 (250μg/mouse, intraperitoneal). At necropsy, small intestines were flushed with cold PBS to count worms. IgG1, IgG2α and mast cell protease-1 (MCPT-1) was measured in serum and supernatants from splenic cells stimulated with ConA (48h) were assessed by ELISA for IL-4, -5, -10 and -13. Doublecortin-like kinase -1 (DCLK-1)+ tuft cells, goblet cells and eosinophils were identified in sections of paraffin embedded or cryopreserved mid-jejunum sections. mRNA of small intestinal epithelium enriched fractions isolated from mice at control and 11 days post-infection (dpi) was evaluated by qPCR. Result(s) Unlike pou2f3+/- and C57Bl6 mice that expel H. diminuta by 8-11 dpi., pou2f3-/- mice harbour H. diminuta at 11 dpi. Pou2f3-/- mice show similar splenic IL-4, -5, -10, -13, and serum IgG1, IgG2a and MCPT-1 levels at 8 dpi. and higher splenic IL-4, -10 at 11 dpi. compared to pou2f3+/- mice. In contrast, pou2f3+/- mice show higher jejunal goblet cell hyperplasia at 8 dpi., higher levels of small intestinal epithelium expression of dclk-1, alox5 and il-25 and jejunal eosinophilia at 11 dpi. compared to pou2f3-/- mice. At 11 dpi, ILCdepletedrag-1-/- develop tuft cell hyperplasia to the same extent as infected PBS/isotype controls and show no trace of intestinal worms in the lumen. Conclusion(s) Enteric tuft cells coordinate rapid expulsion of H. diminuta from mice, but are not essential for the host to detect and develop Th2 systemic responses in response to the worm. However, in the absence of tuft cells, deficiencies are observed in gut-specific effector immune events commonly associated with timely expulsion of enteric helminths; illustrating a disparity between local and systemic immunity following infection with this tapeworm parasite in a non permissive host. The lack of a functional adaptive immune system and surprisingly, depletion of ILC2s, does not completely abrogate worm expulsion or tuft cell hyperplasia in rag-1-/- at 11 dpi. with H. diminuta. This suggests that an innate immune cell population other than CD90.2+ ILC2s amplifies Th2 immunity to H. diminuta-infection. Please acknowledge all funding agencies by checking the applicable boxes below Other Please indicate your source of funding; NSERC, , Eyes High doctoral scholarship, Alberta Graduate excellence scholarship Disclosure of Interest None Declared

Photoperiodic modulation of melatonin receptor and immune genes in migratory redheaded bunting

The transcriptional regulation of innate immune function across annual life history states (LHS) remains obscure in avian migrants. We, therefore, investigated this in a migratory passerine songbird, redheaded bunting (Emberiza bruniceps), which exhibits long-distance vernal migration from India to Central Asia. We exposed the birds (N = 10) to differential photoperiodic conditions to induce a non-migratory (NM), pre-migratory (PM), migratory (MIG), and refractory (REF) state, and performed gene expression assays of melatonin receptors (MEL1A and MEL1B), and innate immunity-linked genes (IL1B, IL6, TLR4, and NFKB) in spleen and blood. We found a significant reduction in splenic mass and volume, and a parallel increase in fat accumulation, and testicular growth in birds under migratory state. The gene expression assay revealed an upregulation of MEL1A and MEL1B mRNA levels in both the tissues in MIG. Additionally, we found a nocturnal increase of splenic IL1B expression, and IL1B, IL6, and TLR4 expression in the blood. The mRNA expression of melatonin receptors and proinflammatory cytokine showed a positive correlation. These results suggest that melatonin relays the photoperiodic signal to peripheral immune organs, which shows LHS-dependent changes in mRNA expression of immune genes.

Resveratrol Attenuates Heat-Stress-Impaired Immune and Inflammatory Responses of Broilers by Modulating Toll-Like Receptor-4 Signaling Pathway

This study investigated the effect of resveratrol on the immune and inflammatory responses and the mRNA levels of splenic toll-like receptor (TLR)-4 signaling pathway-related genes of broilers under heat stress (HS). One hundred and sixty-two birds were allocated to three groups, each with 6 replicates, for 21 continuous days. The three treatments were as follows: the control group (22 ± 1 °C), the HS (33 ± 1 °C for 10 h d-1 and 22 ± 1 °C for the remaining time) group and the HS + resveratrol (400 mg kg-1) group. At the end of the trial, one bird per replicate close to the average body weight (BW) was selected, exsanguinated, and slaughtered. Compared with the control group, the HS treatment decreased (p<0.05) final BW, average daily gain (ADG), average daily feed intake (ADFI), relative weight of bursa of Fabricius and spleen, serum immunoglobulin (Ig) Y, IgA and interleukin (IL)-10 contents, and splenic IL-10 mRNA level, while it increased (p<0.05) feed/gain, mRNA levels of splenic tumor necrosis factor-α (TNF-α), TLR-4, nuclear factor-kappa-B (NF-κB), IL-1β, and IL-6. Compared to the HS group, the HS+resveratrol group exhibited increased (p<0.05) final BW, ADG, relative weight of bursa of Fabricius and spleen, serum IgY, IgA and IL-10 contents, and splenic IL-10 mRNA level, while it exhibited lower (p<0.05) TNF-α, IL-1β and IL-6 contents in serum, and splenic TLR4, TNF-α, IL-1β, and NF-κB mRNA levels. In conclusion, resveratrol prevented a HS-impairment of the immune function of broilers by blocking the abnormal activation of the TLR4 signaling pathway.

Famotidine activates the vagus nerve inflammatory reflex to attenuate cytokine storm

BackgroundSevere COVID-19 is characterized by pro-inflammatory cytokine release syndrome (cytokine storm) which causes high morbidity and mortality. Recent observational and clinical studies suggest famotidine, a histamine 2 receptor (H2R) antagonist widely used to treat gastroesophageal reflux disease, attenuates the clinical course of COVID-19. Because evidence is lacking for a direct antiviral activity of famotidine, a proposed mechanism of action is blocking the effects of histamine released by mast cells. Here we hypothesized that famotidine activates the inflammatory reflex, a brain-integrated vagus nerve mechanism which inhibits inflammation via alpha 7 nicotinic acetylcholine receptor (α7nAChR) signal transduction, to prevent cytokine storm.MethodsThe potential anti-inflammatory effects of famotidine and other H2R antagonists were assessed in mice exposed to lipopolysaccharide (LPS)-induced cytokine storm. As the inflammatory reflex is integrated and can be stimulated in the brain, and H2R antagonists penetrate the blood brain barrier poorly, famotidine was administered by intracerebroventricular (ICV) or intraperitoneal (IP) routes.ResultsFamotidine administered IP significantly reduced serum and splenic LPS-stimulated tumor necrosis factor (TNF) and IL-6 concentrations, significantly improving survival. The effects of ICV famotidine were significantly more potent as compared to the peripheral route. Mice lacking mast cells by genetic deletion also responded to famotidine, indicating the anti-inflammatory effects are not mast cell-dependent. Either bilateral sub-diaphragmatic vagotomy or genetic knock-out of α7nAChR abolished the anti-inflammatory effects of famotidine, indicating the inflammatory reflex as famotidine’s mechanism of action. While the structurally similar H2R antagonist tiotidine displayed equivalent anti-inflammatory activity, the H2R antagonists cimetidine or ranitidine were ineffective even at very high dosages.ConclusionsThese observations reveal a previously unidentified vagus nerve-dependent anti-inflammatory effect of famotidine in the setting of cytokine storm which is not replicated by high dosages of other H2R antagonists in clinical use. Because famotidine is more potent when administered intrathecally, these findings are also consistent with a primarily central nervous system mechanism of action.

Effects of early-life cecal microbiota transplantation from divergently selected inbred chicken lines on growth, gut serotonin, and immune parameters in recipient chickens

Recent studies have revealed that fecal microbiota transplantation exerts beneficial effects on modulating stress-related inflammation and gastrointestinal health of the host. The aim of this study was to examine if cecal microbiota transplantation (CMT) presents similar efficiency in improving the health status of egg-laying strain chickens. Chicken lines 63 and 72 divergently selected for resistance or susceptibility to Marek's disease were used as CMT donors. Eighty-four d-old male recipient chicks (a commercial DeKalb XL layer strain) were randomly assigned into 3 treatments with 7 replicates per treatment and 4 birds per replicate (n = 7): saline (control, CTRL), cecal solution of line 63 (63-CMT), and cecal solution of line 72 (72-CMT) for a 16-wk trial. Cecal transplant gavage was conducted once daily from d 1 to d 10, then boosted once weekly from wk 3 to wk 5. The results indicated that 72-CMT birds had the highest body weight and ileal villus/crypt ratio among the treatments at wk 5 (P ≤ 0.05); and higher heterophil/lymphocyte ratios than that of 63-CMT birds at wk 16 (P < 0.05). 72-CMT birds also had higher levels of plasma natural IgG and Interleukin (IL)-6 at wk 16, while 63-CMT birds had higher concentrations of ileal mucosal secretory IgA at wk 5 and plasma IL-10 at wk 16 (P < 0.05), with a tendency for lower mRNA abundance of splenic IL-6 and tumor necrosis factor (TNF)-α at wk 16 (P = 0.08 and 0.07, respectively). In addition, 72-CMT birds tended to have the lowest serotonin concentrations (P = 0.07) with the highest serotonin turnover in the ileum at wk 5 (P < 0.05). There were no treatment effects on the levels of plasma corticosterone and testosterone at wk 16 (P > 0.05). In conclusion, early postnatal CMT from different donors led to different patterns of growth and health status through the regulation of ileal morphological structures, gut-derived serotonergic activities, peripheral cytokines, and antibody production in recipient chickens.

Effects of Induced Moisture Loss in Chicken Embryos at Embryonic Day 18 and Post-hatch Immune Response During Salmonella enteritidis Lipopolysaccharide Challenge in Broilers.

Two experiments were conducted to investigate the effects of induced moisture loss on embryonic development and the immune response following an inflammatory challenge immediately post-hatch. In Experiment I, fertile leghorn eggs (n = 100) and commercial broiler eggs (n = 300) were set at 37.5°C and moisture loss was induced in one-half of the Leghorn and broiler eggs by drilling two, 1.5 mm diameter holes. The Control eggs had 0 holes. At embryonic day (ED)18, layer and broiler eggs in the 2-holes treatment had a significant (P < 0.01) increase in moisture loss compared to the control treatment (10.1% vs. 8.2%). Similarly, at ED18, the broiler eggs with 2-holes had a significant increase (P < 0.01) in moisture loss compared with control eggs (9.9% vs. 8.4%). Thymocytes from both the leghorn (104%) and broiler (62%) embryos in the 2-holes treatment had significantly increased in vitro proliferation compared with the control embryos (P ≤ 0.05). At ED18, layer and broiler embryos in the 2-holes treatment had an approximate twofold increase in the splenic CD8+/CD4+ ratio (P ≤ 0.05) and CD4+CD25+ cells percentage in both the thymus and spleen (P ≤ 0.05). At ED18, both layer and broiler embryos from the 2-holes treatment had a significant increase in splenic IL1-β, IL-6, IL-10, and TLR-4 mRNA transcription compared to the control group (P ≤ 0.05). Experiment II was repeated with 300 fertile broiler eggs. On the day of hatch, chicks were randomly distributed into one of four treatments in a 2 (0, 2 holes) × 2 (0, 500 μg lipopolysaccharide, LPS) factorial arrangement of treatments. Chicks in the LPS groups were injected intraperitoneally with 500 μg/kg BW LPS. At 24 and 48 h post-hatch, chicks hatched from eggs with 2-holes and challenged with LPS had a significant increase (P ≤ 0.05) in thymocyte proliferation at 24 h (42%) and 48 h (37%) when compared with chicks hatched from the control (0-hole; 0 μg LPS) treatment. Chicks hatched from the 2-holes treatment and challenged with the LPS had an approximately twofold higher splenic CD8+/CD4+ ratio and 1.5 fold increase in CD4+CD25+ percentage compared to control chicks (P ≤ 0.05). In chicks hatched from the 2-holes treatment, MUC2 mRNA transcription was comparable to control chicks at 24 and 48 h in response to the LPS challenge. Our data suggest that the 2-holes treatment reprograms gene transcription to facilitate cell survival via proliferation and differentiation during an LPS inflammatory challenge.

Prenatal Exposure of Rats to Staphylococcal Enterotoxin B Alters the Expression of Th1 and Th2 Cytokines via Decreased Methylation in the Spleen of Adult but not Neonatal Offspring.

The methylation of IFN-γ and IL-4 genes is regarded as an epigenetic regulation that maintains the Th1 or Th2 phenotype. To explore the influence of prenatal administration of the staphylococcal enterotoxin B (SEB) in pregnant rats, on the IFN-γ or IL-4 expression in the offspring spleen. The SEB or PBS was administered intravenously to pregnant rats on the embryo-day 16. After normal delivery, the spleens from the fifth-day neonates and adult offspring were isolated under anesthesia. Quantitative PCR, western blot, ELISA and MeDIP-qPCR were applied to determine the levels of the splenic IFN-γ or IL-4 mRNAs, their protein levels, and methylation status, respectively. Prenatal administration of the SEB in pregnant rats decreased the levels of the splenic IFN-γ and IL-4 proteins in neonates, but increased their mRNA levels. However, prenatal administration of the SEB significantly augmented both mRNA and protein levels of the IFN-γ and IL-4 in the adult spleen. In addition, the prenatal SEB administration decreased the methylation of the splenic IFN-γ and IL-4 in adult but not neonatal offspring. The prenatal administration of SEB in pregnant rats can cause a mixed Th1 and Th2 cytokines response in the offspring spleen, and alter the cytokine expression of the Th1 and Th2 via decreasing the methylation in adult but, not neonatal offspring spleen.

STING Signaling Drives Production of Innate Cytokines, Generation of CD8+ T Cells and Enhanced Protection Against Trypanosoma cruzi Infection.

A variety of signaling pathways are involved in the induction of innate cytokines and CD8+ T cells, which are major players in protection against acute Trypanosoma cruzi infection. Previous data have demonstrated that a TBK-1/IRF3-dependent signaling pathway promotes IFN-β production in response to Trypanosoma cruzi, but the role for STING, a main interactor of these proteins, remained to be addressed. Here, we demonstrated that STING signaling is required for production of IFN-β, IL-6, and IL-12 in response to Trypanosoma cruzi infection and that STING absence negatively impacts activation of IRF-dependent pathways in response to the parasite. We reported no significant activation of IRF-dependent pathways and cytokine expression in RAW264.7 macrophages in response to heat-killed trypomastigotes. In addition, we showed that STING is essential for T. cruzi DNA-mediated induction of IFN-β, IL-6, and IL-12 gene expression in RAW264.7 macrophages. We demonstrated that STING-knockout mice have significantly higher parasitemia from days 5 to 8 of infection and higher heart parasitism at day 13 after infection. Although we observed similar heart inflammatory infiltrates at day 13 after infection, IFN-β, IL-12, CXCL9, IFN-γ, and perforin gene expression were lower in the absence of STING. We also showed an inverse correlation between parasite DNA and the expression of CXCL9, IFN-γ, and perforin genes in the hearts of infected animals at day 13 after infection. Finally, we reported that STING signaling is required for splenic IFN-β and IL-6 expression early after infection and that STING deficiency results in lower numbers of splenic parasite-specific IFN-γ and IFN-γ/perforin-producing CD8+ T cells, indicating a pivotal role for STING signaling in immunity to Trypanosoma cruzi.

Silk Sericin Activates Mild Immune Response and Increases Antibody Production.

To clarify whether nanoparticles of silk sericin (SS) and silk fibroin (SF) can induce inflammation and immune responses, we analyzed splenocyte proliferation, apoptosis and cytokine release to identify the effects of SS and SF on mouse splenocytes in vitro. We implanted mice with SS and SF through intraperitoneal, intramuscular, and subcutaneous routes to evaluate the innate and adaptive immune response to SS and SF in vivo. Cytokines in the serum and spleen were analyzed by Luminex and antibody array. Antigen-specific antibodies were evaluated by enzyme-linked immunosorbent assay (ELISA) at week 1 and 5 after implantation. Distinct cell populations in the spleen and bone marrow were analyzed by flow cytometry. SS suppressed the proliferation of splenocytes and CD11b+CD27- NK cells, induced splenocyte apoptosis, and increased interleukin-1 β (IL-1 β) and tumor necrosis factor-α (TNF-α) in the culture supernatant. SF suppressed splenocyte proliferation, induced splenocyte apoptosis, and increased the titer of TNF-α in culture supernatants. At both week 1 and 5 after implantation with SS, mouse serum interleukin-1 α (IL-1 α) and keratinocyte chemoattractant (KC) were decreased, SS-specific antibody was increased, the proportion of bone marrow CD4+ T cells was increased, and the proportion of splenic neutrophils was decreased. At week 5 after subcutaneous implantation with SF, mouse serum IL-1α, and splenic IL-6, TIMP-1, IL-4, MCP-1, IFN-γ, TCA-3, TNF-α, and IL-17 were decreased. SS was able to induce a mild immune response, as evidenced by CD4+ T cell activation, splenocyte apoptosis, and antigen-specific antibody secretion. Comparatively, SF had low immunogenicity and anti-inflammatory properties.