Effect of Methylmercury Exposure on Bioaccumulation and Nonspecific Immune Respsonses in Hybrid Grouper Epinephelus fuscoguttatus × Epinephelus lanceolatus

Mercury (Hg) is a dangerous heavy metal that can accumulate in fish and is harmful when consumed by humans. This study investigated the bioaccumulation of mercury in the form of methylmercury (MeHg) and evaluated nonspecific immune responses such as phagocytic activity and superoxide anion (O2−) production in hybrid grouper (Epinephelus fuscoguttatus × E. lanceolatus). The hybrid grouper leukocytes were incubated with methylmercury chloride (CH3HgCl) at concentrations of 10–10,000 µg/L to determine cell viability, phagocytic activity, and O2− production in vitro. Subsequently, the grouper were exposed daily to CH3HgCl mixed in the experimental diets at concentrations of 0, 1, 5, and 10 mg/kg for 28 days. The bioaccumulation of MeHg in the liver, head kidney, and muscle tissue was measured, and the phagocytic activity and O2− production were evaluated. In vitro results indicated that cell viability was significantly lower than that of the control group at concentrations > 500 µg/L. The phagocytic rate and O2− production at concentrations ˃ 500 and ˃ 200 µg/L, respectively, were significantly lower than those of the control group. The dietary exposure demonstrated that MeHg accumulated more substantially in the liver and head kidney compared with the muscle tissue in the treatment groups. Moreover, the cumulative concentration significantly increased with higher concentrations and more days of exposure. The phagocytic rate and O2− production in the treatment groups were significantly lower than those in the control group from days 2 and 1, respectively. In conclusion, hybrid grouper accumulated significant MeHg in the liver and head kidney compared with the muscle tissue, and higher concentrations and more exposure days resulted in decreased cell viability, phagocytic activity, and O2− production.

Cooperative phagocytosis underlies macrophage immunotherapy of solid tumours and initiates a broad anti-tumour IgG response

Macrophages are abundant in solid tumours and typically associate with poor prognosis, but macrophage clusters in tumour nests have also been reported as beneficial even though dispersed macrophages would have more contacts with cancer cells. Here, by maximizing both phagocytic activity and macrophage numbers, we discover cooperative phagocytosis by low entropy clusters in rapidly growing engineered immuno-tumouroids. The results fit the calculus of proliferation-versus-engulfment, and rheological measurements and molecular perturbations provide a basis for understanding phagocytic disruption of a tumour's cohesive forces in soft cellular phases. The perturbations underscore the utility of suppressing a macrophage checkpoint in combination with an otherwise ineffective tumour-opsonizing monoclonal antibody, and the approach translates in vivo to tumour elimination that durably protects mice from re-challenge and metastasis. Adoptive transfer of engineered macrophages increases the fraction of mice that eliminate tumours and potentially overcomes checkpoint blockade challenges in solid tumours like insufficient permeation of blocking antibodies and on-target, off-tumour binding. Finally, anti-cancer IgG induced in vivo are tumour-specific but multi-epitope and contribute to a phagocytic feedback that drives macrophage clustering in vitro. Given that solid tumours remain challenging for immunotherapies, durable anti-tumour responses here illustrate unexpected advantages in maximizing net phagocytic activity.

Phagocytic and oxidative burst activity of neutrophils in type 2 diabetic patients with foot ulcers

Introduction and Aim: Diabetic foot ulcers are common complications seen in diabetic patients. Treatment of this disabling foot sore remains a challenge to health care professionals. This study aimed at evaluating whether the neutrophils from type 2 diabetic patients with foot ulcers present an impairment of phagocytic index and impairment in respiratory burst. We also aimed at understanding whether the impairment in neutrophil phagocytic activity can be alleviated with short course of standard treatment regime for foot ulcers. Methodology: For this case-controled study, 43 participants with type 2 diabetes (18 with foot ulcers and 25 without foot ulcers) were prospectively recruited along with 18 healthy volunteers. Phagocytic activity of neutrophils and respiratory burst of neutrophils was assessed along with ESR, percentage neutrophil counts before and after 2 weeks of standard treatment for foot ulcers. Results: Neutrophils of type 2 diabetic patients (with and without foot ulcers ) showed lower levels of phagocytic index and lower percentage of respiratory burst on comparison with non-diabetic subjects. Furthermore, on receiving treatment for foot ulcers, a significant improvement in neutrophil phagocytic indices were observed, along with improvement in wound ulcer score. Conclusion: Phagocytic activity of the neutrophils is impaired in type 2 diabetics (with and without foot ulcers). Neutrophil phagocytic indices can be improved on glycemic control. Additionally, improvement in neutrophil phagocytic indices after short course treatment for foot ulcers can be useful markers to predict treatment efficacy and in prognosis of diabetic foot ulcers.

Sarcodia suieae Acetyl-Xylogalactan Regulates Nile Tilapia (Oreochromis niloticus) Tissue Phagocytotic Activity and Serum Indices

Sarcodia suieae acetyl-xylogalactan was reported to induce macrophage polarisation, and could positively regulate macrophage activation. In this study, we evaluated the effect of Sarcodia suieae acetyl-xylogalactan on the Nile tilapia. First, we assessed the influence of acetyl-xylogalactan on the survival, glucose uptake, and phagocytic activity of tilapia head kidney (THK) melanomacrophage, and observed increased proliferation of these cells in the MTT assay after 12 and 24 h of treatment. Glucose uptake increased in THK melanomacrophage treated with 20 and 30 μg acetyl-xylogalactan for 24 h. Their phagocytic activity was positively enhanced following exposure to acetyl-xylogalactan. Nile tilapia were fed with acetyl-xylogalactan for 4 weeks. At the end of the experiment, Nile tilapia were sacrificed, and the lipopolysaccharide-induced liver and head-kidney apoptosis was examined under reducing conditions in comparison with controls. The phagocytic activities of liver and head-kidney cells were enhanced after 4 weeks of feeding. Blood biochemical analysis revealed a reduction in glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) levels after 4 weeks of feeding. Combined with in vitro and in vivo experiments results, the extracted S. suieae acetyl-xylogalactan could directly induce THK melanomacrophage proliferation, glucose uptake, and phagocytic activity. Acetyl-xylogalactan was able to induce Nile tilapia liver and head-kidney resident macrophage activity, and reduced LPS-induced liver and head-kidney cell apoptosis. S. suieae acetyl-xylogalactan may modulate Nile tilapia macrophage activation by polarising them into M1 macrophages to improve the Nile tilapia nonspecific immune response.

Macrophage Heterogeneity in the Intestinal Cells of Salmon: Hints From Transcriptomic and Imaging Data

The intestine has many types of cells that are present mostly in the epithelium and lamina propria. The importance of the intestinal cells for the mammalian mucosal immune system is well-established. However, there is no in-depth information about many of the intestinal cells in teleosts. In our previous study, we reported that adherent intestinal cells (AIC) predominantly express macrophage-related genes. To gather further evidence that AIC include macrophage-like cells, we compared their phagocytic activity and morphology with those of adherent head kidney cells (AKC), previously characterized as macrophage-like cells. We also compared equally abundant as well as differentially expressed mRNAs and miRNAs between AIC and AKC. AIC had lower phagocytic activity and were larger and more circular than macrophage-like AKC. RNA-Seq data revealed that there were 18309 mRNAs, with 59 miRNAs that were equally abundant between AIC and AKC. Integrative analysis of the mRNA and miRNA transcriptomes revealed macrophage heterogeneity in both AIC and AKC. In addition, analysis of AIC and AKC transcriptomes revealed functional characteristics of mucosal and systemic macrophages. Five pairs with significant negative correlations between miRNA and mRNAs were linked to macrophages and epithelial cells and their interaction could be pointing to macrophage activation and differentiation. The potential macrophage markers suggested in this study should be investigated under different immune conditions to understand the exact macrophage phenotypes.

TRPA1-mediated effects on the functional activity of macrophages under the exposure with cigarette smoke and cinnamaldehyde

Introduction. Being the leading cause of COPD, smoking represents a major health problem. Upon entering the respiratory tract, cigarette smoke comes into contact with various cells, including macrophages expressing on their surface TRPA1 receptors, which are sensitive to the main pathogenic compounds formed during tobacco combustion.Aim. To study the functional activity of TRPA1 channels on macrophages in terms of cell responses to cigarette smoke and the TRPA1 agonist cinnamaldehyde (CA). Materials and methods. The experimental conditions included exposure of monocyte-derived macrophages to CA (100 μM), 4% cigarette smoke extract (CSE) and 4% CSE after pretreatment with TRPA1 selective antagonist (HC-030031 100 μM). The concentration of cytokines in the culture medium, the expression of TRPA1 on the cell surface, as well as the phagocytic activity of macrophages were analyzed by flow cytometry.Results. We found that 60.2 (49.6; 71.8)% of cells expressed TRPA1 and their number increased after exposure with CA. CSE significantly inhibited CXCL10 production from 1121.3 (295.7; 3154.6) pg/ml to 187.9 (113.8; 398.3) pg/ml (p=0.04), which was partially prevented by blocking TRPA1 (692.4 [428.6; 2916.6] pg/ml, p=0.04). Similar to CSE, CA also caused a decrease in CXCL10 concentration (189.2 [111.7; 311.3] pg/ml, p=0.03). Among other observations, there was an increase in the concentration of IL-1β after the exposition with HC-030031, as well as a decrease in TNF-α, IFN-γ and IL-12p70 after the treatment with CA. CSE caused a minor inhibition in phagocytic cells number, which was not prevented by TRPA1 blocking. CA, on the contrary, increased the phagocytic activity of macrophages. The initial expression of TRPA1 had a negative correlation with the dynamics of CXCL10 in response to CSE and CA but a positive correlation with the number of phagocytic cells after exposition with CA (ρ=0.81, p=0.005). Conclusions. TRPA1 expressed on macrophages apparently mediate an anti-inflammatory effect in terms of produced cytokines but increase phagocytic activity of the cells. TRPA1 are also major receptors involved in the diminished CXCL10 production by macrophage under exposition with cigarette smoke

Functional Ex Vivo Testing of Alveolar Monocytes in Patients with Pneumonia-Related ARDS

Biomarkers of disease severity might help with individualizing the management of patients with acute respiratory distress syndrome (ARDS). During sepsis, a sustained decreased expression of the antigen-presenting molecule human leucocyte antigen-DR (HLA-DR) on circulating monocytes is used as a surrogate marker of immune failure. This study aimed at assessing whether HLA-DR expression on alveolar monocytes in the setting of a severe lung infection is associated with their functional alterations. BAL fluid and blood from immunocompetent patients with pneumonia-related ARDS admitted between 2016 and 2018 were isolated in a prospective monocentric study. Alveolar and blood monocytes were immunophenotyped using flow cytometry. Functional tests were performed on alveolar and blood monocytes after in vitro lipopolysaccharide (LPS) stimulation. Phagocytosis activity and intracellular tumor necrosis factor (TNF) production were quantified using fluorochrome-conjugated-specific antibodies. Ten ARDS and seven non-ARDS control patients were included. Patients with pneumonia-related ARDS exhibited significantly lower HLA-DR expression both on circulating (p < 0.0001) and alveolar (p = 0.0002) monocytes. There was no statistically significant difference observed between patient groups (ARDS vs. non-ARDS) regarding both alveolar and blood monocytes phagocytosis activity. After LPS stimulation, alveolar (p = 0.027) and blood (p = 0.005) monocytes from pneumonia-related ARDS patients had a significantly lower intracellular TNF expression than non-ARDS patients. Monocytes from pneumonia-related ARDS patients have a deactivated status and an impaired TNF production capacity but display potent phagocytic activity. HLA-DR level expression should not be used as a surrogate marker of the phagocytic activity or the TNF production capacity of alveolar monocytes.

A unique NLRC4 receptor from echinoderms mediates Vibrio phagocytosis via rearrangement of the cytoskeleton and polymerization of F-actin

Many members of the nucleotide-binding and oligomerization domain (NACHT)- and leucine-rich-repeat-containing protein (NLR) family play crucial roles in pathogen recognition and innate immune response regulation. In our previous work, a unique and Vibrio splendidus-inducible NLRC4 receptor comprising Ig and NACHT domains was identified from the sea cucumber Apostichopus japonicus, and this receptor lacked the CARD and LRR domains that are typical of common cytoplasmic NLRs. To better understand the functional role of AjNLRC4, we confirmed that AjNLRC4 was a bona fide membrane PRR with two transmembrane structures. AjNLRC4 was able to directly bind microbes and polysaccharides via its extracellular Ig domain and agglutinate a variety of microbes in a Ca2+-dependent manner. Knockdown of AjNLRC4 by RNA interference and blockade of AjNLRC4 by antibodies in coelomocytes both could significantly inhibit the phagocytic activity and elimination of V. splendidus. Conversely, overexpression of AjNLRC4 enhanced the phagocytic activity of V. splendidus, and this effect could be specifically blocked by treatment with the actin-mediated endocytosis inhibitor cytochalasin D but not other endocytosis inhibitors. Moreover, AjNLRC4-mediated phagocytic activity was dependent on the interaction between the intracellular domain of AjNLRC4 and the β-actin protein and further regulated the Arp2/3 complex to mediate the rearrangement of the cytoskeleton and the polymerization of F-actin. V. splendidus was found to be colocalized with lysosomes in coelomocytes, and the bacterial quantities were increased after injection of chloroquine, a lysosome inhibitor. Collectively, these results suggested that AjNLRC4 served as a novel membrane PRR in mediating coelomocyte phagocytosis and further clearing intracellular Vibrio through the AjNLRC4-β-actin-Arp2/3 complex-lysosome pathway.

The effect of the drug “Amiksyn” on the immune status of carp

The productivity of pond fish, in particular carp, largely depends on the intensity of use of aquatic bioresources. The aim of the study was to investigate the effect of “Amiksyn” on the immunological parameters of carp fish, namely: the number of T- and B-lymphocytes, immunoglobulins, the level of circulating immune complexes, lysozyme, phagocytic activity and bactericidal activity of serum. 10 fish were selected for hematological and biochemical studies. The research was conducted in the aquarium of the Department of Aquatic Bioresources of Stepan Gzhytskyi NUVMB Lviv. Fish were kept in the pool for 21 days to adapt to the new conditions, and at the beginning of the series of experiments were placed in aquariums with a volume of 200 liters. Aeration and mechanical filtration of water were provided in the tanks. The temperature during the experiments fluctuated slightly and was 18 ± 1.5 °C. The main hydrochemical parameters corresponded to fish farming standards. In order to adjust the immune response, and thus prevent possible manifestations of fish damage by pathogens, we studied the possibility of using the immunostimulant “Amiksyn”, which was used for 5 – 10 – 15 – 20 days and then dosing 5 – 10 – 15 mg/kg body weight Pisces. The use of “Amiksyn” at a dose of 10 mg/kg of fish weight four times during the fifteen-day period with an interval of 5 days provided a decrease in the number of T-lymphocytes by 9.1 % and B-lymphocytes by 23.0 %; caused the activation of non-specific resistance indicators, namely lysozyme activity increased by 28.7 %, phagocytic activity of leukocytes increased by 14.0 % and bactericidal activity of serum increased by 19.0 %. Therefore, to adjust the immune system and stimulate the metabolic processes of fish in order to prevent the negative effects of various pathogens, the most optimal dose was 10 mg/kg of fish weight within 15 days of use.