Platelet Toll-Like Receptor 4–Related Innate Immunity Potentially Participates in Transfusion Reactions Independent of ABO Compatibility: An Ex Vivo Study

The role of platelet TLR4 in transfusion reactions remains unclear. This study analyzed platelet TLR4 and certain damage-associated molecular patterns (DAMPs) and evaluated how ABO compatibility affected TLR4 expression after a simulated ex vivo transfusion. A blood bank was the source of donor red blood cells. Blood from patients undergoing cardiac surgery was processed to generate a washed platelet suspension to which the donor blood was added in concentrations 1, 5, and 10% (v/v). Blood-mixing experiments were performed on four groups: a 0.9% saline control group (n = 31); a matched-blood-type mixing group (group M, n = 20); an uncross-matched ABO-specific mixing group (group S, n = 20); and an ABO-incompatible blood mixing group (group I, n = 20). TLR4 expression in the platelets was determined after blood mixing. We evaluated levels of TLR4-binding DAMPs (HMGB1, S100A8, S100A9, and SAA), lipopolysaccharide-binding protein, and endpoint proteins in the TLR4 signaling pathway. In the M, S, and I groups, 1, 5, and 10% blood mixtures significantly increased TLR4 expression (all p < 0.001) in a concentration-dependent manner. Groups M, S, and I were not discovered to have significantly differing TLR4 expression (p = 0.148). HMGB1, S100A8, and S100A9 levels were elevated in response to blood mixing, but SAA, lipopolysaccharide-binding protein, TNF-α, IL-1β, and IL-6 levels were not. Blood mixing may elicit innate immune responses by upregulating platelet TLR4 and DAMPs unassociated with ABO compatibility, suggesting that innate immunity through TLR4-mediated signaling may induce transfusion reactions.

Remimazolam Protects Against LPS-Induced Endotoxicity Improving Survival of Endotoxemia Mice

Remimazolam is a new benzodiazepine of sedative drugs with an ultra-short-acting anesthetic effect, commonly used for critically ill patients (especially septic patients) in intensive care units (ICUs). Although some anesthetics have been reported to show certain anti-inflammatory effects, the role of remimazolam in inflammation is still remained unknown. Here, we studied the effects of remimazolam on macrophage in response to LPS both in vivo and in vitro. Interestingly, compared with LPS treatment group, remimazolam remarkably improved survival rate of endotoxemia mice and decreased the release of LPS-induced inflammatory mediators (such as TNF-α, IL-6, and IL-1β). We further found that remimazolam not only inhibited the activation of MAPK signal pathway at 15 min after LPS treatment but also disturbed Rab5a related TLR4 expression at cell surface in response to LPS at a later time. Such evidence suggests that remimazolam might be beneficial to septic patients who are suffering from uncontrolled inflammatory responses.

MiR-140-5p inhibits morphine tolerance in rats by targeting TLR4

Purpose: To determine the influence of miR-140-5p on morphine tolerance in rats.Methods: Sprague-Dawley (SD) rats were randomly divided into morphine tolerance (MT) and saline control (NS) groups, respectively. Rats in MT group were injected with 10 μL (10 μg) morphine twice daily for seven consecutive days while those in NS group were administered the equivalent volume of normal saline. The maximum effect of morphine (MPE) was computed from tail-flick test results. MiR-140-5p mimics and toll-like receptor 4 (TLR4) lentivirus were transfected separately or co-transfected into model rats. MiR-140-5p and TLR4 expression were determined by quantitative real-time polymerase chain reaction (RT-qPCR) or western blotting. Dual-luciferase reporter assay was used to verify the target relationship between miR-140-5p and TLR4.Results: The expression of miR-140-5p was decreased, while the expression of TLR4 increased in morphine-tolerant rats (p < 0.05). TLR4 was a target of miR-140-5p. At 24 and 48 h after injection, MPE clearly increased and TLR4 expression was reduced under miR-140-5p overexpression or TLR4 knockdown (p < 0.05). Moreover, there were no significant changes in MPE or levels of TLR4 when miR-140-5p and TLR4 were co-transfected into morphine-tolerant rats.Conclusion: MiR-140-5p inhibits morphine resistance in rats via targeted regulation of TLR4 expression. These provide a theoretical basis for the clinical management of morphine tolerance.

Immunohistochemical Analysis of Inflammasome Signaling Component Expression Among Patients with Low Risk and High Risk Myelodysplastic Syndrome Using Tissue Microarray

Background: Myelodysplastic syndromes (MDS) are characterized by aberrant maturation, ineffective hematopoiesis, cytopenias, and progression to acute myeloid leukemia. MDS pathogenesis is multifactorial and potentially linked to constitutive innate immune stimulation converging upon the NLRP3 inflammasome to induce pyroptosis, a caspase-1 dependent cell death. Inflammasome assembly is initiated by both cell-extrinsic stimuli including S100A9, the TLR4 and CD33 ligand, and cell-intrinsic danger signals licensing caspase-1 which activates IL1b and beta-catenin resulting in cell death and increased cellular proliferation leading to maturation and differentiation blocks. Further, EYA2 has been suggested to be an inflammasome activator, whereas PLA2 has been suggested to be an inhibitor. Recent work demonstrates immunohistochemistry (IHC) may be utilized to assess expression of inflammasome components. The purpose of the present study is to compare inflammasome component expression among patients with low and high risk MDS and correlate these findings with clinical outcome data. Methods: An IRB protocol was approved prior to initiating this study. We retrospectively identified 87 patients with MDS including 40 (46%) low risk MDS and 47 (54%) high risk MDS patients. A tissue microarray (TMA) was constructed utilizing MDS bone marrow biopsy samples (2-3 representative cores per sample). IL-1, S100A9, EYA2, CPLA2, Beta Catenin, and TLR4 expression were assessed by IHC after validation of each antibody. IHC expression was scored independently by two hematopathologists by calculating scores (product of staining intensity x percent expression). IHC expression was compared using Spearman correlation estimate. Demographic and clinical data were collected and correlated with IHC expression using Kruskal-Wallis test, Spearman correlation, and Logrank test. Results: Patients were median 72 years of age, 69% men and included 29% MDS-EB2, 28% MDS-MLD, 17% MDS-RS, 15% MDS-EB1, 9% MDS-SLD, 1% MDS del5q and 1% MDS/MPN-U. Correlation matrix in Figure 1 illustrates correlation seen between expression of various inflammasome components. Increased expression of CPLA2 and TLR4 (median 0.6 and 1.71, respectively) were observed in high risk MDS compared to low risk MDS (median 0.12 and 0.02, respectively, p&lt;0.0001), and decreased beta-catenin expression was seen in high risk MDS compared to low risk MDS (median 0.00 vs 0.14, p&lt;0.0001). IL-1, S100A9, and EYA2 expression was similar between high risk MDS and low risk MDS samples (median score 1.55 vs 1.55, 0.56 vs 0.60, and 0.74 vs 0.60, respectively). Simple linear regression demonstrates relationship between R-IPSS and CPLA2 (r 2=0.175, p&lt;0.001), beta-catenin (r 2=-0.182, p&lt;0.001), and TLR4 (r 2=0.555, p&lt;0.001). Progression free survival (112.5 months vs 19.8 months) and overall survival (107.6 months vs 24.5 months) were longer for low risk MDS compared to high risk MDS, respectively (p&lt;0.0001). Overall survival was increased in patients with low CPLA2 expression compared to high CPLA2 expression (88.9 months vs 38.1 months, p =0.0018), high beta-catenin expression compared to low beta-catenin expression (130.0 months vs 36.8 months, p &lt; 0.0001) and low TLR4 expression compared to high TLR4 expression (90.1 months vs 22.3 months, p &lt; 0.001). Conclusions: IHC staining of inflammasome activators using TMA may allow better characterization of molecular pathways contributing the MDS pathogenesis. A correlation was seen between expression of antigens known to be increased downstream of NLRP3 inflammasome activation. Survival was increased in patients with low CPLA2 expression, high beta-catenin expression, and low TLR4 expression. Further characterization of inflammasome signaling may lead to novel therapeutic targets and prognostic tools in the management of MDS. Figure 1 Figure 1. Disclosures Lancet: Millenium Pharma/Takeda: Consultancy; Celgene/BMS: Consultancy; ElevateBio Management: Consultancy; BerGenBio: Consultancy; AbbVie: Consultancy; Daiichi Sankyo: Consultancy; Agios: Consultancy; Astellas: Consultancy; Jazz: Consultancy. Komrokji: Jazz: Consultancy, Speakers Bureau; AbbVie: Consultancy; Acceleron: Consultancy; Geron: Consultancy; Taiho Oncology: Membership on an entity's Board of Directors or advisory committees; PharmaEssentia: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; BMSCelgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Estrogen Promotes Pituitary Prolactinoma by Upregulating TLR4 / NF-κB/p38MAPK Pathway

Background: Prolactinomas have harmful effects on human health, and the pathogenesis is still unknown. Furthermore, the morbidity of women is much more than man, maybe related with estradiol level. Thus, it is important to reveal the pathogenesis and develop new therapeutic methods for prolactinomas.Methods: Immunofluorescence analysis or Immunohistochemistry analysis were performed on the ERβ, TLR4 and prolactin (PRL) expressions of pituitary gland in C57BL/6 mice and human prolactinoma specimen. In the present study, the role of TLR4 in prolactinoma was determined using estradiol-induced mice models in C57BL/6 wild-type (WT) and TLR4−/− mice. MMQ cells were treated with estradiol, fulvestrant, LPS or transfected with different TLR4 small interfering RNA, which to study ERβ, TLR4 and PRL expression in MMQ cells. Co‑immunoprecipitates analysis was used to investigate the interaction between ERβ and TLR4.Results: Immunofluorescence analysis or Immunohistochemistry analysis showed that PRL and TLR4 expression were co-located and increased in the pituitary gland of mice and human prolactinoma specimen compared with the control specimen. It was shown that PRL and TLR4 expression was co-located and increased significantly in the pituitary gland of estradiol-injected prolactinoma mice compared with the control mice. Knockout of TLR4 significantly inhibited tumor overgrowth, and PRL expression was decreased in estradiol-induced mice through regulating TLR4/NF‑κB/p38MAPK pathway. Estradiol promoted PRL expression through regulating TLR4/NF‑κB/p38MAPK pathway in vitro study, and pre-Inhibiting ERβ or TLR4 reverse the effect, while simultaneously activating ERβ and TLR4 enhanced PRL expression than activating single ERβ or TLR4. Furthermore, ERβ co-immunoprecipitates with endogenous TLR4 was assessed by co-immunoprecipitation analysis.Conclusions: These results suggest that estradiol promoted prolactinoma development by activating the TLR4/NF‑κB/ p38MAPK pathway through Erβ and TLR4 knockout inhibited the proliferation and secretion of prolactin in prolactinoma.

Epithelial-Specific TLR4 Knockout Challenges Current Evidence of TLR4 Homeostatic Control of Gut Permeability

<b><i>Introduction:</i></b> Toll-like receptor 4 (TLR4) is a highly conserved immunosurveillance protein of innate immunity, displaying well-established roles in homeostasis and intestinal inflammation. Current evidence shows complex relationships between TLR4 activation, maintenance of health, and disease progression; however, it commonly overlooks the importance of site-specific TLR4 expression. This omission has the potential to influence translation of results as previous evidence shows the differing and distinct roles that TLR4 exhibits are dependent on its spatiotemporal expression. <b><i>Methods:</i></b> An intestinal epithelial TLR4 conditional knockout (KO) mouse line (<i>Tlr4</i>^<i>ΔIEC</i>, <i>n</i> = 6–8) was utilized to dissect the contribution of epithelial TLR4 expression to intestinal homeostasis with comparisons to wild-type (WT) (<i>n</i> = 5–7) counterparts. Functions of the intestinal barrier in the ileum and colon were assessed with tissue resistance in Ussing chambers. Molecular and structural comparisons in the ileum and colon were assessed via histological staining, expression of tight junction proteins (occludin and zonular occludin 1 [ZO-1]), and presence of CD11b-positive immune cells. <b><i>Results:</i></b> There was no impact of the intestinal epithelial TLR4 KO, with no differences in (1) tissue resistance–ileum (mean ± standard error of mean [SEM]): WT 22 ± 7.2 versus <i>Tlr4</i>^<i>ΔIEC</i> 20 ± 5.6 (Ω × cm²) <i>p</i> = 0.831, colon WT 30.8 ± 3.6 versus <i>Tlr4</i>^<i>ΔIEC</i> 45.1 ± 9.5 <i>p</i> = 0.191; (2) histological staining (overall tissue structure); and (3) tight junction protein expression (% area stain, mean ± SEM)–ZO-1: ileum–WT 1.49 ± 0.155 versus <i>Tlr4</i>^<i>ΔIEC</i> 1.17 ± 0.07, <i>p</i> = 0.09; colon–WT 1.36 ± 0.26 versus <i>Tlr4</i>^<i>ΔIEC</i> 1.12 ± 0.18 <i>p</i> = 0.47; occludin: ileum–WT 1.07 ± 0.12 versus <i>Tlr4</i>^<i>ΔIEC</i> 0.95 ± 0.13, <i>p</i> = 0.53; colon–WT 1.26 ± 0.26 versus <i>Tlr4</i>^<i>ΔIEC</i> 1.02 ± 0.16 <i>p</i> = 0.45. CD11b-positive immune cells (% area stain, mean ± SEM) in the ileum were mildly decreased in WT mice: WT 0.14 ± 0.02 versus <i>Tlr4</i>^<i>ΔIEC</i> 0.09 ± 0.01 <i>p</i> = 0.04. However, in the colon, there was no difference in CD11b-positive immune cells between strains: WT 0.53 ± 0.08 versus <i>Tlr4</i>^<i>ΔIEC</i> 0.49 ± 0.08 <i>p</i> = 0.73. <b><i>Conclusions:</i></b> These data have 2 important implications. First, these data refute the assumption that epithelial TLR4 exerts physiological control of intestinal physiology and immunity in health. Second, and most importantly, these data support the use of the <i>Tlr4</i>^<i>ΔIEC</i> line in future models interrogating health and disease, confirming no confounding effects of genetic manipulation.

The Influence of Infection by Different Leishmania (Viannia) braziliensis Isolates on the Pathogenesis of Disseminated Leishmaniasis

Disseminated Leishmaniasis (DL) is an emerging and severe form of Leishmania (Viannia) braziliensis infection defined by the presence of 10 and up to more than 1,000 skin lesions. The mechanisms underlying parasite dissemination remain unknown. Genotypic differences among species of L. braziliensis have been associated with different clinical forms of disease. The present work compared the function of monocytes obtained from patients with cutaneous leishmaniasis (CL) and DL in response to infection with L. braziliensis isolates of both these two clinical forms of disease. Mononuclear cells obtained from DL and CL patients were infected with different L. braziliensis isolates, and numbers of infected cells, parasite load, respiratory burst, TLR2 and TLR4 expression and cytokine production were evaluated. DL isolates infected more monocytes, induced greater respiratory burst, and more cytokine production compared to isolates from CL patients regardless of the origin of monocytes (DL or CL). However, greater parasite multiplication and higher TLR2 and TLR4 expression were seen in monocytes from DL patients compared to CL following infection with DL isolates. Our results indicate the participation of both parasite genotype and host factors in the pathogenesis of DL.

Prognostic Value of NLRP3 Inflammasome and TLR4 Expression in Breast Cancer Patients

Inflammasome complexes play a pivotal role in different cancer types. NOD-like receptor protein 3 (NLRP3) inflammasome is one of the most well-studied inflammasomes. Activation of the NLRP3 inflammasome induces abnormal secretion of soluble cytokines, generating advantageous inflammatory surroundings that support tumor growth. The expression levels of the NLRP3, PYCARD and TLR4 were determined by immunohistochemistry in a cohort of primary invasive breast carcinomas (BCs). We observed different NLRP3 and PYCARD expressions in non-tumor vs tumor areas (p&lt;0.0001). All the proteins were associated to more aggressive clinicopathological characteristics (tumor size, grade, tumor proliferative activity etc.). Univariate analyses were carried out and related Kaplan-Meier curves plotted for NLRP3, PYCARD and TLR4 expression. Patients with higher NLRP3 and TLR4 expression had worse 5-year disease-free survival (DFS) compared to patients with lower NLRP3 and TLR4 expression (p =0.021 and p = 0.009, respectively). In multivariate analysis, TLR4 was confirmed as independent prognostic factors for DFS (HR = 2.03, 95% CI 1.16–3.57, p = 0.014), and high NLRP3 expression showed a slight association with DFS (HR = 1.75, 95% CI 0.98–3.15, p = 0.06). In conclusion, we showed TLR4 expression as independent prognostic factors and we highlighted for the first time that high expression of NLRP3 is linked to a poor prognosis in BC patients. These results suggest that NLRP3 and TLR4 could be two new good prognostic factor for BC patients.

Melatonin as an immunomodulator in children with Down syndrome

Background Down syndrome (DS) is a disorder characterised by marked immune dysfunction, increased mortality from sepsis, chronic inflammation, increased oxidative stress, sleep disturbance and possibly abnormal endogenous melatonin levels. Melatonin has a myriad of immune functions, and we hypothesised that this therapeutic agent could modulate the innate immune system in this cohort. Methods We investigated neutrophil and monocyte function (CD11b, TLR4 expression by flow cytometry), genes involved in TLR signalling (MyD88, IRAK4, TRIF), the inflammasome (NLRP3, IL-1β), and circadian rhythm (BMAL, CLOCK, CRY) by qPCR, and inflammatory cytokines (IL-2, IL-6, IL-8, IL-18, IL-1β, TNF-α, IFN-γ, IL-10, IL-1ra, VEGF, Epo, GM-CSF) by enzyme-linked immunosorbent assay (ELISA) following immunomodulation with LPS endotoxin and melatonin. 47 children with DS and 23 age- and sex-matched controls were recruited. Results We demonstrated that melatonin has several significant effects by reducing CD11b and TLR4 expression, attenuating TLR signalling, genes involved in the inflammasome and has the potential to reduce LPS-induced inflammatory responses. Conclusions Immunomodulatory effects of melatonin were found in both paediatric cohorts with more marked effects in the children with DS. Melatonin mediates immune response through a wide array of mechanisms and this immunomodulator may buffer the inflammatory response by regulating pro and anti-inflammatory signalling. Impact We highlight that melatonin mediates its immune response through a wide array of mechanisms, its effects appear to be dose dependant and children with Down syndrome may be more receptive to treatment with it. Immunomodulatory effects of melatonin were demonstrated with marked effects in the children with Down syndrome with a reduction of MyD88, IL-1ß and NLRP3 expression in whole-blood samples. Melatonin is a proposed anti-inflammatory agent with a well-established safety profile, that has the potential for mitigation of pro- and anti-inflammatory cytokines in paediatric Down syndrome cohorts, though further clinical trials are warranted.

Microbiome diversity declines while distinct expansions of Th17, iNKT, and dendritic cell subpopulations emerge after anastomosis surgery

Background Anastomotic failure causes morbidity and mortality even in technically correct anastomoses. Initial leaks must be prevented by mucosal reapproximation across the anastomosis. Healing is a concerted effort between intestinal epithelial cells (IECs), immune cells, and commensal bacteria. IEC TLR4 activation and signaling is required for mucosal healing, leading to inflammatory factor release that recruits immune cells to limit bacteria invasion. TLR4 absence leads to mucosal damage from loss in epithelial proliferation, attenuated inflammatory response, and bacteria translocation. We hypothesize after anastomosis, an imbalance in microbiota will occur due to a decrease in TLR4 expression and will lead to changes in the immune milieu. Results We isolated fecal content and small intestinal leukocytes from murine, Roux-en-Y and end-to-end anastomoses, to identify microbiome changes and subsequent alterations in the regulatory and pro-inflammatory immune cells 3 days post-operative. TLR4+ IECs were impaired after anastomosis. Microbiome diversity was reduced, with Firmicutes, Bacteroidetes, and Saccharibacteria decreased and Proteobacteria increased. A distinct TCRβhi CD4+ T cells subset after anastomosis was 10–20-fold greater than in control mice. 84% were Th17 IL-17A/F+ IL-22+ and/or TNFα+. iNKT cells were increased and TCRβhi. 75% were iNKT IL-10+ and 13% iNKTh17 IL-22+. Additionally, Treg IL-10+ and IL-22+ cells were increased. A novel dendritic cell subset was identified in anastomotic regions that was CD11bhi CD103mid and was 93% IL-10+. Conclusions This anastomotic study demonstrated a decrease in IEC TLR4 expression and microbiome diversity which then coincided with increased expansion of regulatory and pro-inflammatory immune cells and cytokines. Defining the anastomotic mucosal environment could help inform innovative therapeutics to target excessive pro-inflammatory invasion and microbiome imbalance.