Increased Expression Of Cytokines Research Articles

METTL3 Regulates Osteoblast Differentiation and Inflammatory Response via Smad Signaling and MAPK Signaling.

Osteoblasts are crucial bone-building cells that maintain bone homeostasis, whereas inflammatory stimuli can inhibit osteogenesis and activate inflammatory response. N6-methyladenosine (m6A) is the most abundant mRNA modification in eukaryotes and plays important roles in multiple biological processes. However, whether m6A modification affects osteoblast differentiation and inflammatory response remains unknown. To address this issue, we investigated the expression of the N6-adenosine methyltransferase METTL3 and found that it was upregulated during osteoblast differentiation and downregulated after LPS stimulation. We then knocked down METTL3 and observed decreased levels of osteogenic markers, ALP activity, and mineralized nodules, as well as Smad1/5/9 phosphorylation, in LPS-induced inflammation. METTL3 knockdown promoted the mRNA expression and stability of negative regulators of Smad signaling, Smad7 and Smurf1, the same regulatory pattern identified when the m6A-binding protein YTHDF2 was silenced. Moreover, METTL3 depletion enhanced proinflammatory cytokine expression and increased the phosphorylation of ERK, p38, JNK, and p65 in MAPK and NF-κB signaling pathways. The increase in cytokine expression was inhibited after MAPK signaling inhibitor treatment. All data suggest that METTL3 knockdown inhibits osteoblast differentiation and Smad-dependent signaling by stabilizing Smad7 and Smurf1 mRNA transcripts via YTHDF2 involvement and activates the inflammatory response by regulating MAPK signaling in LPS-induced inflammation.

Lipopolysaccharide-induced inflammation leads to acute elevations in pro-inflammatory cytokine expression in a mouse model of Fragile X syndrome

Fragile X syndrome (FXS) is a neurodevelopmental disorder caused by a single genetic mutation in the Fmr1 gene, serving as the largest genetic cause of intellectual disability. Trinucleotide expansion mutations in Fmr1 result in silencing and hypermethylation of the gene, preventing synthesis of the RNA binding protein Fragile X mental retardation protein which functions as a translational repressor. Abnormal immune responses have been demonstrated to play a role in FXS pathophysiology, however, whether these alterations impact how those with FXS respond to an immune insult behaviorally is not entirely known. In the current study, we examine how Fmr1 knockout (KO) and wild type (WT) mice respond to the innate immune stimulus lipopolysaccharide (LPS), both on a molecular and behavioral level, to determine if Fmr1 mutations impact the normal physiological response to an immune insult. In response to LPS, Fmr1 KO mice had elevated hippocampal IL-1β and IL-6 mRNA levels 4 h post-treatment compared to WT mice, with no differences detected in any cytokines at baseline or between genotypes 24 h post-LPS administration. Fmr1 KO mice also had upregulated hippocampal BDNF gene expression 4 h post-treatment compared to WT mice, which was not dependent on LPS administration. There were no differences in hippocampal protein expression between genotypes in microglia (Iba1) or astrocyte (GFAP) reactivity. Further, both genotypes displayed the typical sickness response following LPS stimulation, demonstrated by a significant reduction in food burrowed by LPS-treated mice in a burrowing task. Additional investigation is critical to determine if the transient increases in cytokine expression could lead to long-term changes in downstream molecular signaling in FXS.

Understanding the pathogenesis of Flavobacterium psychrophilum using the rainbow trout monocyte/macrophage-like cell line, RTS11, as an infection model

The life cycle of Flavobacterium psychrophilum (Fp), the causative agent of bacterial coldwater disease (BCWD) and rainbow trout fry syndrome (RTFS), appears to involve interactions with spleen and head kidney macrophages. To develop an in vitro model for studying this, F. psychrophilum was incubated with a rainbow trout splenic monocyte/macrophage-like cell line (RTS11) and fundamental macrophage functions evaluated. The animal cell basal medium, L15, supplemented with bovine serum (FBS) supports RTS11 maintenance, and surprisingly, L15 with 2% FBS (L15/FBS) also supported F. psychrophilum growth. L15/FBS in which the bacteria had been grown is referred to as F. psychrophilum conditioned medium (FpCM). Adding FpCM to RTS11 cultures caused a small, yet significant, percentage of cells to die, many cells to become more diffuse, and phagocytosis to be temporarily reduced. FpCM also significantly stimulated transcript expression for pro-inflammatory cytokines (IL-1β, TNFα and IL-6) and the anti-inflammatory cytokine (IL-10) after one day of exposure but this upregulation rapidly declined over time. Adding live F. psychrophilum to RTS11 cultures also altered the cellular morphology and stimulated cytokine expression more profoundly than FpCM. Additionally, the phagocytic activity of RTS11 was also significantly impaired by live F. psychrophilum, but not to the same extent as when exposed to FpCM. Adding heat-killed bacteria to RTS11 cultures elicited few changes. These bacteria/RTS11 co-cultures should be useful for gaining a deeper understanding of the pathogenesis of F. psychrophilum and may aid in the development of effective measures to prevent infection and spread of this troublesome disease.

The Role of TTP Phosphorylation in the Regulation of Inflammatory Cytokine Production by MK2/3.

Tristetraprolin (TTP) is an RNA-binding protein and an essential factor of posttranscriptional repression of cytokine biosynthesis in macrophages. Its activity is temporally inhibited by LPS-induced p38MAPK/MAPKAPK2/3-mediated phosphorylation, leading to a rapid increase in cytokine expression. We compared TTP expression and cytokine production in mouse bone marrow-derived macrophages of different genotypes: wild type, MAPKAP kinase 2 (MK2) deletion (MK2 knockout [KO]), MK2/3 double deletion (MK2/3 double KO [DKO]), TTP-S52A-S178A (TTPaa) knock-in, as well as combined MK2 KO/TTPaa and MK2/3 DKO/TTPaa. The comparisons reveal that MK2/3 are the only LPS-induced kinases for S52 and S178 of TTP and the role of MK2 and MK3 in the regulation of TNF biosynthesis is not restricted to phosphorylation of TTP at S52/S178 but includes independent processes, which could involve other TTP phosphorylations (such as S316) or other substrates of MK2/3 or p38MAPK Furthermore, we found differences in the dependence of various cytokines on the cooperation between MK2/3 deletion and TTP mutation ex vivo. In the cecal ligation and puncture model of systemic inflammation, a dramatic decrease of cytokine production in MK2/3 DKO, TTPaa, and DKO/TTPaa mice compared with wild-type animals is observed, thus confirming the role of the MK2/3/TTP signaling axis in cytokine production also in vivo. These findings improve our understanding of this signaling axis and could be of future relevance in the treatment of inflammation.

Membrane trafficking as an active regulator of constitutively secreted cytokines.

Immune-cell activation by inflammatory stimuli triggers the transcription and translation of large amounts of cytokines. The transport of newly synthesized cytokines to the plasma membrane by vesicular trafficking can be rate-limiting for the production of these cytokines, and immune cells upregulate their exocytic machinery concomitantly with increased cytokine expression in order to cope with the increasing demand for trafficking. Whereas it is logical that trafficking is rate-limiting for regulated secretion where an intracellular pool of molecules is waiting to be released, the reason for this is not obvious for constitutively secreted cytokines, such as interleukin-6 (IL-6), interleukin-12 (IL-12) and tumor necrosis factor-α (TNF-α). These constitutively secreted cytokines are primarily regulated at the transcriptional and/or translational level but mounting evidence presented here shows that cells might also increase or decrease the rate of post-Golgi cytokine trafficking to modulate their production. Therefore, in this Hypothesis, we ask the question: why is there a need to limit the trafficking of constitutively secreted cytokines? We propose a model where cells monitor and adjust their production rate of cytokines by sensing the intracellular level of cytokines while they are in transit to the plasma membrane. This self-regulation of cytokine production could prevent an overshooting response of acute-phase cytokines, such as IL-6, IL-12 and TNF-α, upon acute infection.

TIGIT expression is upregulated in T cells and causes T cell dysfunction independent of PD-1 and Tim-3 in adult B lineage acute lymphoblastic leukemia

T cell immunoglobulin and ITIM domain (TIGIT) is a novel immune checkpoint receptor and plays critical roles in cancer immunity. Adult acute lymphoblastic leukemia (ALL) remains a treatment challenge despite years of research. In this study, we analyzed the status of TIGIT expression in circulating T cells from patients with adult ALL. Compared to the data in healthy controls, the expression of TIGIT in CD4+CD25− T cells and CD8+ T cells in adult ALL patients presented a small but significant upregulation. Stimulation via the CD3/CD28 route increased TIGIT mRNA expression at 24 h, which peaked at 48 h and was maintained at 72 h post-stimulation. The frequency of TIGIT+ cells, on the other hand, consistently increased over time. ALL protein lysate or Wilms’ Tumor 1 peptide could significantly increase the expression of TIGIT in ALL, but not healthy control T cells. Compared to TIGIT− cells, the TIGIT+ cells presented significantly higher PD-1 and Tim-3 expression directly ex vivo, and significantly lower IL-2, IFN-γ, and TNF-α after CD3/CD28 stimulation. The high inhibitory molecule and low cytokine expression signature was especially pronounced in ALL TIGIT+ CD4+CD25− T cells and TIGIT+ CD8+ T cells. Blocking TIGIT alone could minimally increase cytokine expression independent of PD-1 and Tim-3 blocking, whereas blocking TIGIT, PD-1, and Tim-3 altogether was significantly more effective. Together, these data demonstrated that TIGIT regulated T cell function in adult ALL patients, and may serve as a treatment target for ALL.

320.8: Identification of biomarkers for risk, diagnosis, and prognosis of GVHD after bowel transplantation and central role of graft resident memory T cells in pathogenesis

Introduction: Graft-versus-host disease (GVHD) is a relatively common and highly morbid complication after intestinal transplantation. Its pathophysiology remains poorly understood. Resident memory T cells (TRM) are a newly described T cell subset with memory phenotype localizing to peripheral tissue. We hypothesized that the pathophysiology of GVHD might be related to increased donor TRM in the graft that subsequently migrate into host blood and tissue. Methods: Intestinal transplantation from deceased donors was performed using our standard method. Graft and blood lymphocytes from 10 patients with GVHD and 34 without were longitudinally analyzed using flow cytometry. Results: Levels of CD4 and CD8 TRM were approximately 20% higher in the grafts of GVHD vs. stable patients prior to implantation and significantly higher at the time of GVHD (p = 0.02 and 0.04). There was also a mean 60.3% higher level of CD8 TRM in the native bowels of GVHD patients compared to controls and 20–30% higher levels of IFN-γ and TNF-α expression in both the grafts and native bowels of GVHD patients. The percentage of CD4 and CD8 TRM in the blood of GVHD vs. stable patients significantly increased during GVHD (p = 0.005), and expression of HLA-DR, CD57, and PD-1 was significantly higher. There were also significant increases in CD8 effector memory cells (p = 0.0056) and decreases in naïve cells (p = 0.0034). Notably, CD8/PD-1 was also significantly elevated prior to transplantation in patients who later had GVHD (p = 0.025), and a pre-transplant CD8/PD1 level >45% was an absolute predictor of later GVHD. Moreover, we found significantly higher percentages of HLA-DR, CD57, and PD-1 in patients with GVHD who died vs. those who survived, and values that always correlated with mortality were CD8/CD57 > 35%, CD4/HLA-DR > 15%, CD8/HLA-DR > 45%, and CD4/PD1 > 35%. Conclusion: In the largest longitudinal analysis to date, we demonstrate that increased TRM percentage and inflammatory cytokine expression in graft bowel correspond with increased TRM in blood and native bowel as well as increased cytokine expression in native bowel at time of GVHD. Thus GVHD pathogenesis may depend on donor TRM in graft bowel migrating to the blood and native tissue of recipients. Recipients with higher PD-1 expression, indicating T cell exhaustion, might be more vulnerable, providing a possible biomarker for GVHD risk, while increased expression of maturity and activation markers correlate with prognosis.

Abstract 2339: High fat diet modulates host immunity and impairs the efficacy of combined radiation and immunotherapy

Abstract Introduction: Obesity is associated with a higher incidence of cancer, increased cancer mortality, impaired responses to radiation therapy (RT), and worse toxicity after RT. Paradoxically, recent studies suggest that obese patients may actually have improved outcomes with immunotherapy (IO), such as programmed death (PD)-1 inhibition, highlighting a link between the host’s metabolic state and immune homeostasis. We therefore hypothesized that a high fat diet could be used to alter the host’s immune response to improve the efficacy of combined RT and IO. Materials/Methods: 12-week old female Balb/c mice were injected with 5x104 syngeneic, luciferase tagged, triple-negative, murine breast cancer cells (4T1). Upon development of a palpable tumor, the mice were randomized to either an ad lib (AL) or high fat (HF), defined as 45% calories from fat, diet. Additionally, each cohort was treated with either diet alone (D), diet plus RT (4 Gy in 3 daily fractions), diet plus PD-1 inhibitor, or a combination of all three (Combo). On day 26, the mice were injected with luciferin and tumor growth was measured using the IVIS bioluminescence in vivo imaging system. At time of sacrifice, primary tumors and spleen were harvested for downstream analysis of immune markers via RNA microarray and flow cytometry. Results: The median overall survival (OS) for animals fed either an HF or AL diet was 36 and 39 days, respectively (NS). The difference in OS however became much more apparent between the individual treatment arms: HF+αPD1 vs AL+αPD1 (37d vs 41.5d, p=0.041); HF+RT vs AL+RT (36d vs 45d, p=0.039); HF+Combo vs AL+Combo (39d vs 48d, p=0.037). HF fed mice also developed metastases sooner than AL fed mice across all arms (D: 22d vs 30d; RT: 24d vs 32d; αPD1: 24d vs 34d; Combo: 27d vs 37d, p=0.047). Microarray analysis of primary tumor tissue revealed an at least 1.5 fold increase expression of cytokines associated with inflammation including IFN-y, TNF-α, IL-17F, IL-22 and IL-1β in the HF diet groups while AL fed mice demonstrated increased expression of the anti-inflammatory cytokines IL-10 and TGF-β. Consistent with the increase in inflammatory markers after a HF diet, flow cytometric analysis of splenocytes revealed a significant decrease in the number of CD4+CD25+Foxp3+ regulatory T cells in the HF group compared with AL fed mice (3.3% versus 7.68%, respectively; p = 0.006). Notably, these Tregs also produced less IL-10 compared with those from AL mice (24.4% vs 54.5%, p=0.037). Conclusions: We demonstrate that a HF diet impairs the efficacy of RT, IO, and combined therapy. This is associated with the development of a pro-inflammatory state demonstrated by increased expression of pro-inflammatory cytokines and a decrease in the number and activity of regulatory T cells. Based on these findings, we conclude that diet can directly impact treatment efficacy and should be considered as a variable in future clinical studies. Note: This abstract was not presented at the meeting. Citation Format: Gregor Manukian, Brittany A. Simone, Tiziana DeAngelis, Kevin Ko, Charles Kivolowitz, Sylvester Jusu, Nicole L. Simone. High fat diet modulates host immunity and impairs the efficacy of combined radiation and immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2339.

TOX and TOX2 transcription factors cooperate with NR4A transcription factors to impose CD8+ T cell exhaustion

T cells expressing chimeric antigen receptors (CAR T cells) have shown impressive therapeutic efficacy against leukemias and lymphomas. However, they have not been as effective against solid tumors because they become hyporesponsive ("exhausted" or "dysfunctional") within the tumor microenvironment, with decreased cytokine production and increased expression of several inhibitory surface receptors. Here we define a transcriptional network that mediates CD8+ T cell exhaustion. We show that the high-mobility group (HMG)-box transcription factors TOX and TOX2, as well as members of the NR4A family of nuclear receptors, are targets of the calcium/calcineurin-regulated transcription factor NFAT, even in the absence of its partner AP-1 (FOS-JUN). Using a previously established CAR T cell model, we show that TOX and TOX2 are highly induced in CD8+ CAR+ PD-1high TIM3high ("exhausted") tumor-infiltrating lymphocytes (CAR TILs), and CAR TILs deficient in both TOX and TOX2 (Tox DKO) are more effective than wild-type (WT), TOX-deficient, or TOX2-deficient CAR TILs in suppressing tumor growth and prolonging survival of tumor-bearing mice. Like NR4A-deficient CAR TILs, Tox DKO CAR TILs show increased cytokine expression, decreased expression of inhibitory receptors, and increased accessibility of regions enriched for motifs that bind activation-associated nuclear factor κB (NFκB) and basic region-leucine zipper (bZIP) transcription factors. These data indicate that Tox and Nr4a transcription factors are critical for the transcriptional program of CD8+ T cell exhaustion downstream of NFAT. We provide evidence for positive regulation of NR4A by TOX and of TOX by NR4A, and suggest that disruption of TOX and NR4A expression or activity could be promising strategies for cancer immunotherapy.

Human Induced Pluripotent Stem Cell–Derived Cardiomyocyte Patch in Rats With Heart Failure

To treat chronic heart failure (CHF), we developed a robust, easy to handle bioabsorbable tissue-engineered patch embedded with human neonatal fibroblasts and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). This patch was implanted on the epicardial surface of the heart covering the previously infarcted tissue. Sprague-Dawley rats (6-8 weeks old) underwent sham surgery (n= 12) or left coronary artery ligation (n= 45). CHF rats were randomized 3 weeks after ligation to CHF control with sham thoracotomy (n= 21), or a fibroblasts/hiPSC-CMs patch (n= 24) was implanted. All sham surgery rats also underwent a sham thoracotomy. At 3 weeks after randomization, hemodynamics, echocardiography, electrophysiologic, and cell survival studies were performed. Patch-treated rats had decreased (P < .05) left ventricular-end diastolic pressure and the time constant of left ventricular relaxation (Tau), increased anterior wall thickness in diastole, and improved echocardiography-derived indices of diastolic function (E/e' [ratio of early peak flow velocity to early peak LV velocity] and e'/a' [ratio of early to late peak left ventricular velocity]). All rats remained in normal sinus rhythm, with no dysrhythmias. Rats treated with the patch showed improved electrical activity. Transplanted hiPSC-CMs were present at 7 days but not detected at 21 days after implantation. The patch increased (P < .05) gene expression of vascular endothelial growth factor, angiopoietin 1, gap junction α-1 protein (connexin 43), β-myosin heavy 7, and insulin growth factor-1 expression in the infarcted heart. Epicardial implantation of a fibroblasts/hiPSC-CMs patch electrically enhanced conduction, lowered left ventricular end-diastolic pressure, and improved diastolic function in rats with CHF. These changes were associated with increases in cytokine expression.

SUN-051 Increased Circulating Cytokines in African American Women with Obesity and Elevated HbA1c

Background: The leading cause of death in US women is cardiovascular disease (CVD). Obesity and type-2 diabetes (T2D) are independent risk factors for cardiovascular disease while obesity is the leading risk factor for T2D. CDC reports that 53% of African American women are obese, compared to 32% of European American women. They also suggest that the prevalence of obesity may contribute to the increase in prevalence of diabetes; 85.2% of people with T2D are overweight or obese. The CDC estimates that 1 in 3 Americans will have diabetes by 2050, if this trend continues. Obesity, diabetes, and CVD are all associated with inflammation created by the expansion of adipose tissue. Low grade inflammation is associated with insulin resistance and precedes the onset of T2D in adults. Methods: A total of 30 serum samples were collected from African American women: 15 obese, normal HbA1c and 15 obese, high HbA1c. Clinical metabolic parameters were determined using standard commercial kits administered by Laboratory Corporation of America. Thirty chemokines/cytokines were measured using Luminex X-MAP® technology. Cytokine levels between the two groups were compared using unpaired t-test, followed by Pearson’s correlation coefficient (r) to determine correlation between cytokine expression and clinical metabolic parameters. Results: Serum levels of IL-3 (P=0.007), IL-4 (P=0.02), IL-7 (P=0.002), TNF-α (P=0.006), IFN-α2 (P=0.001) and CX3CL1 (P=0.02) were significantly higher in obese, African American women with elevated HbA1c. IL-7 (R=0.53, P=0.01) and TNF-α (R=0.37, P=0.04) were positively correlated with HbA1c in all patients. In patients with elevated HbA1c, IL-3 (R=-0.72, P=0.005) and IFN-α2 (R=-0.83, P=0.05) were negatively correlated with HbA1c. Several clinical metabolic parameters were also correlated with cytokines. TNF- α (R=0.48, P=0.008), IL-7 (R=0.58, P=0.005), CX3CL1 (R=0.50, P=0.006) and IFN- α2 (R=0.42, P=0.03) were positively correlated with triglycerides in all patients. TNF- α (R=0.49, P=0.007), IL-7 (R=0.58, P=0.005), CX3CL1 (R=0.50, P=0.006) and IFN- α2 (R=0.42, P=0.03) were positively correlated with VLDL in all patients. In patients with elevated HbA1c, IL-7 (R=-0.65, P=0.03) and IFN- α2 (R=-0.57, P=0.04) were negatively correlated with HDL. IL-7 (R=-0.58, P=0.005) was negatively correlated with HDL in all patients. Conclusions: Increased cytokine expression of IL-3, IL-4, IL-7, TNF- α, IFN- α2, and CX3CL1 is associated with T2D in obese, African American women. Circulating cytokine expression is associated with increased cardio-metabolic disease risk. Our observations provide insights into T2D and propose new avenues for therapeutic strategies to combat obesity and its associated metabolic abnormalities.

A New Understanding of the Role of IL-1 in Age-Related Intervertebral Disc Degeneration in a Murine Model.

Increased cytokine expression, in particular interleukin-1β (IL-1β), is considered a hallmark of intervertebral disc degeneration. However, the causative relationship between IL-1 and age-dependent degeneration has not been established. To investigate the role of IL-1 in driving age-related disc degeneration, we studied the spine phenotype of global IL-1α/β double knockout (IL-1KO) mice at 12 and 20 months. Multiplex ELISA analysis of blood revealed significant reductions in the concentrations of IFN-γ, IL-5, IL-15, TNF-α, IP-10, and a trend of reduced concentrations of IL-10, macrophage inflammatory protein 1α (MIP-1α), keratinocyte chemoattractant/human growth-regulated oncogene (KC/GRO), and IL-6. However, the circulating level of MIP-2, a neutrophil chemoattractant, was increased in the IL-1KO. The alterations in systemic cytokine levels coincided with altered bone morphology-IL-1KO mice exhibited significantly thicker caudal cortical bone at 12 and 20 months. Despite these systemic inflammatory and bony changes, IL-1 deletion only minimally affected disc health. Both wild-type (WT) and IL-1KO mice showed age-dependent disc degeneration. Unexpectedly, rather than protecting the animals from degeneration, the aging phenotype was more pronounced in IL-1KO animals: knockout mice evidenced significantly more degenerative changes in the annulus fibrosis (AF) together with alterations in collagen type and maturity. At 20 months, there were no changes in nucleus pulposus (NP) extracellular matrix composition or cellular marker expression; however, the IL-1KO NP cells occupied a smaller proportion of the NP compartment that those of WT controls. Taken together, these results show that IL-1 deletion altered the systemic inflammatory environment and vertebral bone morphology. However, instead of protecting discs from age-related disc degeneration, global IL-1 deletion amplified the degenerative phenotype. © 2019 American Society for Bone and Mineral Research.

Alpha-momorcharin regulates cytokine expression and induces apoptosis in monocytes

Background and aim: Alpha-momorcharin (α-MMC) is a type I ribosome-inactivating protein (RIP) that is purified from Momordica charantia. Despite its strong antitumor activities, α-MMC exerts the undesirable immunotoxicity effects of hypersensitivity or immunosuppression. Since α-MMC is a plant protein, its application in vivo can easily induce hypersensitivity, but its immunosuppressive mechanism is still unclear.Materials and methods: The toxicity of α-MMC to peripheral blood cells and the cytokine expression in peripheral blood mononuclear cells (PBMCs) and spleen immune cells were measured in rats. For further confirmation, experiments were performed in vitro with the mononuclear cell line THP-1, B lymphocyte cell line WIL2-S and T lymphocyte cell line Jurkat.Results: High doses of α-MMC (3.0 mg/kg) resulted in weight loss in rats, a decreased percentage of monocytes, and increased percentages of eosinophils and basophils. Both high-dose and low-dose (1.0 mg/kg) α-MMC inhibited cytokine expression in PBMCs and increased cytokine expression in spleen T cells. In in vitro, α-MMC mainly acted on THP-1 cells, with effects including high dose-induced apoptosis and low dose-induced regulation of inhibitory cytokine expression.Conclusions: The action of α-MMC on immune cells mainly affects monocytes, thereby eliciting its immunosuppressive effect. Its mode of action is to guide functional immunosuppressive regulation at low doses and induce apoptosis at high doses. As the monocytes would be recruited into tumor tissues and are polarized into tumor-associated macrophages, the selective cytotoxicity and cytokine release regulation of α-MMC in monocytes may be an important mechanism of its antitumor effects.

Exposure to in utero inflammation increases locomotor activity, alters cognitive performance and drives vulnerability to cognitive performance deficits after acute immune activation

Fetal exposure to intrauterine inflammation (IUI) affects brain development. Using intrauterine lipopolysaccharide (LPS) administration to induce a localized, rather than a systemic, inflammation, we have previously shown that IUI increases cytokine expression and microglia number, and reduces white matter in the brains of exposed offspring. Clinical data suggest that IUI may increase the risk for cognitive and neurodevelopmental disorders, however, IUI is often found in the context of preterm birth, making it difficult to disentangle the adverse effects of inflammation from those related to prematurity. Therefore, using a mouse model of IUI that does not involve preterm birth, operant tasks were used to evaluate motivation, attention, impulsivity, and locomotion. IUI-exposed offspring were found to have increased locomotion and increased motivation (females only), and testing in the 5-choice serial reaction time task (5-CSRTT) showed that IUI-exposed offspring performed more trials and could respond accurately at a shorter stimulus length. We have previously shown that IUI animals have a potentiated cytokine response to a “second hit” (acute LPS injection) in adulthood, so animals’ performance in the 5CSRTT was evaluated following an acute injection of LPS. As opposed to the improved performance observed under baseline conditions, IUI exposed animals demonstrated a greater decrease in performance after an acute LPS administration. To identify putative molecular mechanisms underlying this potentiated decline in cognitive performance, PFC samples were collected immediately after post-LPS cognitive testing and targeted gene expression analysis was correlated with specific measures of cognitive performance. Three receptors important for neuron-microglia crosstalk were found to correlate with task performance in the males following acute LPS administration. These data demonstrate that early life exposure to localized inflammation of the uterus, in the absence of prematurity, increases locomotor activity and improves some aspects of cognitive performance, but drives a vulnerability for adult cognitive performance deficits in response to acute infection.

Effects of palmitate and astaxanthin on cell viability and proinflammatory characteristics of mesenchymal stem cells

Mesenchymal stem cells (MSCs) have broad immunomodulatory activities. These cells are a stable source of cytokine production such as interleukin-6 (IL6), monocyte chemoattractant protein-1 (MCP-1/CCL2) and vascular endothelial growth factor (VEGF). Fatty acid elevation in chronic metabolic diseases alters the microenvironment of MSCs and thereby, might affect their survival and cytokine production. In the present study, we investigated the effects of palmitate, the most abundant saturated free fatty acid (FFA) in plasma, and astaxanthin, a potent antioxidant, on cell viability and apoptosis in human bone marrow-driven mesenchymal stem cells. We also elucidated how palmitate and astaxanthin influence the inflammation in MSCs. Human mesenchymal stem cells were collected from an aspirate of the femurs and tibias marrow compartment. The effect of palmitate on cell viability, caspase activity and pro-inflammatory cytokines expression and secretion were evaluated. In addition, activation of the MAP kinases and NF-kB signaling pathways were investigated. The results showed that astaxanthin protected MSCs from palmitate-induced cell death. We found that palmitate significantly enhanced IL-6, VEGF and MCP-1 expression, and secretion in MSC cells. Increased cytokine expression was parallel to the enhanced phosphorylation of P38, ERK and IKKα-IKKβ. In addition, pretreatment with JNK, ERK, P38, and NF-kB inhibitors could correspondingly attenuate palmitate-induced expression of VEGF, IL-6, and MCP-1. Our results demonstrated that fatty acid exposure causes inflammatory responses in MSCs that can be alleviated favorably by astaxanthin treatment.

RAMP1 in Kupffer cells is a critical regulator in immune-mediated hepatitis.

The significance of the relationship between the nervous and immune systems with respect to disease course is increasingly apparent. Immune cells in the liver and spleen are responsible for the development of acute liver injury, yet the regulatory mechanisms of the interactions remain elusive. Calcitonin gene-related peptide (CGRP), which is released from the sensory nervous system, regulates innate immune activation via receptor activity-modifying protein 1 (RAMP1), a subunit of the CGRP receptor. Here, we show that RAMP1 in Kupffer cells (KCs) plays a critical role in the etiology of immune-mediated hepatitis. RAMP1-deficient mice with concanavalin A (ConA)-mediated hepatitis, characterized by severe liver injury accompanied by infiltration of immune cells and increased secretion of pro-inflammatory cytokines by KCs and splenic T cells, showed poor survival. Removing KCs ameliorated liver damage, while depleting T cells or splenectomy led to partial amelioration. Adoptive transfer of splenic T cells from RAMP1-deficient mice led to a modest increase in liver injury. Co-culture of KCs with splenic T cells led to increased cytokine expression by both cells in a RAMP1-dependent manner. Thus, immune-mediated hepatitis develops via crosstalk between immune cells. RAMP1 in KCs is a key regulator of immune responses.

Duck innate immune responses to high and low pathogenicity H5 avian influenza viruses

Ducks are the reservoir host of influenza A viruses, and are permissive for replication of most strains, yet can elicit robust innate immune responses to highly pathogenic strains. Tissue tropism and viral amino acid differences affect virulence, but we have limited knowledge about how viral differences influence the host innate immune response. Here we compare the innate immune response in Pekin ducks to a recombinant highly-pathogenic avian influenza (HPAI) H5N1 virus and a naturally arising attenuated variant of this strain that differs at one amino acid in polymerase A (T515A), as well as ducks infected with two different H5 strains of low pathogenic avian influenza (LPAI). Using qPCR we examined the relative abundance of transcripts for RIG-I and interferon-beta (IFNβ), and downstream interferon stimulated genes (ISGs). The polymerase PA (T515A) mutation did not significantly affect replication in vivo but greatly attenuated host interferon responses. ISG induction was robust for both H5N1 strains, but was three times lower for the PA mutant strain. Low pathogenic viruses elicited detectable induction of RIG-I, IFNβ and ISGs in lung and intestine tissues that correlated with the recovery of viruses from tracheal or cloacal swabs. Several genes in the MAVS signaling pathway were also upregulated by H5N1, which contributed to further amplification of the signal. We also examined hematoxylin-eosin stained tissue sections and observe evidence of lung pathology and splenocyte depletion with both H5N1 viruses at 3 dpi, and recovery by 6 dpi. However, for both H5N1 strains we observed inflammation around neurons in brain, with increased cytokine expression in some individuals. Our findings reveal HPAI H5N1 viruses induced stronger innate immune responses to the infection, while LPAI viruses elicit a milder response.

Candida albicans induces TLR2/MyD88/NF-κB signaling and inflammation in oral lichen planus-derived keratinocytes.

The risk of oral lichen planus (OLP), a chronic inflammatory oral mucosal disease, becoming malignant increases by 21-fold in patients with fungal infection. This study examined the impact of Candida albicans exposure on Toll-like receptor (TLR) signaling in primary keratinocyte cultures obtained from OLP patients. Following co-culture of primary OLP keratinocyte cultures with C. albicans for 24 hours, inflammatory cytokine concentrations were determined by ELISA. TLR2, MyD88, and NF-κBp65 mRNA and protein expression were assessed using quantitative RT-PCR and Western blot analyses, respectively. Keratinocyte apoptosis was also determined by flow cytometry. IL-10, IL-8, IL-2, and TNF-ɑ levels were significantly higher following co-culture with C. albicans (all p ≤ 0.034). MyD88, NF-κB p65, and TLR2 mRNA (all p < 0.001) and protein (all p ≤ 0.004) expression levels were significantly higher in OLP keratinocytes following C. albicans exposure. Finally, the apoptosis rates of OLP keratinocytes were 21.2%, 29.4%, and 25.4% for the control cells and 3.9%, 5.6%, and 4.4% for those exposed to C. albicans, suggesting that co-culture with C. albicans inhibits the apoptosis of OLP keratinocytes. C. albicans activates the TLR2/MyD88/NF-κB signaling pathway in OLP keratinocytes, resulting in increased cytokine expression and decreased keratinocyte apoptosis. Two key events in the pathogenesis of OLP and its progression to malignancy, namely increased inflammation and decreased apoptosis, were induced by exposure to C. albicans. Thus, targeting this signaling pathway may represent a novel therapeutic strategy to prevent OLP malignant transformation.

Abstract 411: Human Species-specific Loss of the CMP-N-acetylneuraminic Acid Hydroxylase Fuels Atherosclerosis Development via Intrinsic and Extrinsic Mechanisms

Cardiovascular disease (CVD) events due to atherosclerosis are very common in humans, but rarely occur spontaneously in other mammals, absent experimental manipulation. All humans exhibit a species-specific deficiency of the common mammalian sialic acid N-glycolylneuraminic acid (Neu5Gc), due to pseudogenization of the CMP-N-acetylneuraminic acid (Neu5Ac) hydroxylase (CMAH) gene, which occurred in hominin ancestors about 2-3 million years ago. Human-like Cmah -/- mice that express only the precursor sialic acid Neu5Ac are more prone to insulin resistance and have more reactive macrophages and T cells. Human dietary consumption of Neu5Gc (primarily from red meat), can acts as a foreign “xenoantigen” in humans that gets metabolically incorporated into endogenous glycoproteins. Humans with circulating anti-Neu5Gc “xeno-autoantibodies” can thus potentially develop local chronic inflammation or “xenosialitis” at sites of Neu5Gc accumulation such as endothelial cells and in atherosclerotic plaques. In this study we set out to test if human CMAH deficiency contributes to CVD via multiple intrinsic and extrinsic mechanisms. Cmah -/- Ldlr -/- mice had increased atherogenesis on a Neu5Gc-free high fat diet (HFD), compared to Cmah +/+ Ldlr -/- mice. This was not associated with cytokine levels in plasma, but increased cytokine expression was seen in Cmah -/- Ldlr -/- macrophages in comparison to Cmah +/+ Ldlr -/- . The baseline relative hyperglycemia of the Cmah -/- Ldlr -/- mice was also enhanced on a Neu5Gc-free HFD. When such mice were immunized to develop human-like levels of anti-Neu5Gc antibodies, they had a 2.5-fold increase in atherosclerosis on a Neu5Gc-rich HFD compared to control Neu5Ac-rich or sialic acid-free HFD feeding. Drastically advanced lesions with increased necrotic core areas and infiltration of macrophages and T-cells accompanied increases in atherosclerotic area and lesion volume. None of these differences were explained by changes in lipoprotein profiles or insulin sensing. Human evolutionary loss of CMAH likely contributes to atherosclerosis propensity, via both intrinsic mechanisms such as amplified chronic inflammatory response and hyperglycemia; and extrinsic mechanisms such as red meat-derived Neu5Gc-induced xenosialitis.

Effects of treadmill training and EVOO enriched diet on cartilage degeneration in rats with ACLT.

The beneficial effects of Extra Virgin Olive Oil (EVOO), the main fat source in Mediterranean diet, are widely studied thanks to its anti-inflammatory and antioxidant properties. Lubricin is a chondroprotective glycoprotein, with lubricant properties. A joint injury causes an increased cytokine expression, associated with decreased lubricin synthesis and predisposition to cartilage degeneration. The aim of this study was to evaluate the beneficial role of EVOO-enriched diet and physical activity, on osteoarthritic cartilage of rats. These effects were assessed through lubricin and interleukin-6 expression in rat joint tissues. Osteoarthritis was induced mechanically by anterior cruciate ligament transection (ACLT). Forty-eight 3-month-old male Wistar rats were used for this study. Experiments and surgical procedures for ACLT were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee (I.A.C.U.C.) of the University of Catania, European Community Council Directive and the Italian Animal Protection Law (Protocol n. 2112015-PR, Italian Ministry of Health). The 48 animals were divided into 6 groups: 1 – control rats (no ACLT, common die); 2 – rats with ACLT and common diet (experimental control); 3 – rats with ACLT, common diet and treadmill training; 4 – rats with ACLT, Sicilian EVOO and treadmill training; 5 – rats with ACLT, Tunisian EVOO and treadmill training; 6 – rats with ACLT, Tunisian EVOO and leaves extract and treadmill training. To this purpose, we performed histomorphometric, histological, immunocytochemical, immunohistochemical and biochemical analysis for lubricin and interleukin-6 evaluations in articular cartilage and synovial fluid of rats. Furthermore, skeletal muscle (anterior tibial) and liver tissue were used to perform histological analyses to exclude respectively hepatic steatosis and sarcopenia. The results showed the beneficial effect of physical activity and EVOO supplementation on the rat articular cartilage. ACLT determined an increase in interleukin-6 expression and a significant decrease in the lubricin expression, while physical activity and EVOO supplemented diet, determined that the values returned to a normal level when compared to the control group. Moreover, EVOO does not cause hepatic steatosis and muscle fibers of all groups did not show damaged histological structure. Our findings suggest that mechanical stimulation is able to increase the release of lubricin in articular cartilage, while the anti-inflammatory properties of EVOO reduces the expression of interleukin-6. In conclusion, the results showed a beneficial effect of the conjunction of EVOO-based diet, corresponding to the Mediterranean diet, and physical activity on the preservation of articular cartilage tissue. Support or Funding Information This study was supported by the University Research Project Grant (Triennial Research Plan 2016–2018), University of Catania, Italy. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.