Neutrophil Diversity (Immature, Aged, and Low-Density Neutrophils) and Functional Plasticity: Possible Impacts of Iron Overload in ß-Thalassemia.

Neutrophil dysfunction is a form of immune suppression in patients with ß-thalassemia (Beta-thal), although data on this are limited. In this study, blood from patients and healthy volunteers was analyzed. Flow cytometry analysis demonstrated an increase in immature neutrophils (CD16- CD62L+) and aged (senescent) neutrophils (CD16+ CD62L-) in Beta-thal patients compared to healthy volunteers. The Beta-thal neutrophils demonstrated less prominent chemotaxis and phagocytosis than healthy neutrophils at the baseline. With phorbol myristate acetate (PMA) or lipopolysaccharide (LPS) stimulations, some of the indicators, including the flow cytometry markers (CD11b, CD62L, CD66b, CD63, apoptosis, and reactive oxygen species) and neutrophil extracellular traps (NETs; detected by anti-citrullinated histone 3 immunofluorescence), were lower than the control. Additionally, low-density neutrophils (LDNs), which are found in the peripheral blood mononuclear cell (PBMC) fraction, were observed in Beta-thal patients but not in the control group. The expression of CD11b, CD66b, CD63, arginase I, and ROS in LDNs was higher than the regular normal-density neutrophils (NDNs). The proliferation rate of CD3+ T cells isolated from the PBMC fraction of healthy volunteers was higher than that of the cells from patients with Beta-thal. The incubation of red blood cell (RBC) lysate plus ferric ions with healthy NDNs transformed the NDNs into the aged neutrophils (decreased CD62L) and LDNs. In conclusion, iron overload induces neutrophil diversity along with some dysfunctions.

The Regulatory Roles of Ezh2 in Response to Lipopolysaccharide (LPS) in Macrophages and Mice with Conditional Ezh2 Deletion with LysM-Cre System.

The responses of macrophages to lipopolysaccharide (LPS) might determine the direction of clinical manifestations of sepsis, which is the immune response against severe infection. Meanwhile, the enhancer of zeste homologue 2 (Ezh2), a histone lysine methyltransferase of epigenetic regulation, might interfere with LPS response. Transcriptomic analysis on LPS-activated wild-type macrophages demonstrated an alteration of several epigenetic enzymes. Although the Ezh2-silencing macrophages (RAW264.7), using small interfering RNA (siRNA), indicated a non-different response to the control cells after a single LPS stimulation, the Ezh2-reducing cells demonstrated a less severe LPS tolerance, after two LPS stimulations, as determined by the higher supernatant TNF-α. With a single LPS stimulation, Ezh2 null (Ezh2flox/flox; LysM-Crecre/-) macrophages demonstrated lower supernatant TNF-α than Ezh2 control (Ezh2fl/fl; LysM-Cre-/-), perhaps due to an upregulation of Socs3, which is a suppressor of cytokine signaling 3, due to the loss of the Ezh2 gene. In LPS tolerance, Ezh2 null macrophages indicated higher supernatant TNF-α and IL-6 than the control, supporting an impact of the loss of the Ezh2 inhibitory gene. In parallel, Ezh2 null mice demonstrated lower serum TNF-α and IL-6 than the control mice after an LPS injection, indicating a less severe LPS-induced hyper-inflammation in Ezh2 null mice. On the other hand, there were similar serum cytokines after LPS tolerance and the non-reduction of serum cytokines after the second dose of LPS, indicating less severe LPS tolerance in Ezh2 null mice compared with control mice. In conclusion, an absence of Ezh2 in macrophages resulted in less severe LPS-induced inflammation, as indicated by low serum cytokines, with less severe LPS tolerance, as demonstrated by higher cytokine production, partly through the upregulated Socs3.

Less Severe Polymicrobial Sepsis in Conditional mgmt-Deleted Mice Using LysM-Cre System, Impacts of DNA Methylation and MGMT Inhibitor in Sepsis.

The O6-methylguanine-DNA methyltransferase (MGMT) is a DNA suicide repair enzyme that might be important during sepsis but has never been explored. Then, the proteomic analysis of lipopolysaccharide (LPS)-stimulated wild-type (WT) macrophages increased proteasome proteins and reduced oxidative phosphorylation proteins compared with control, possibly related to cell injury. With LPS stimulation, mgmt null (mgmtflox/flox; LysM-Crecre/-) macrophages demonstrated less profound inflammation; supernatant cytokines (TNF-α, IL-6, and IL-10) and pro-inflammatory genes (iNOS and IL-1ß), with higher DNA break (phosphohistone H2AX) and cell-free DNA, but not malondialdehyde (the oxidative stress), compared with the littermate control (mgmtflox/flox; LysM-Cre-/-). In parallel, mgmt null mice (MGMT loss only in the myeloid cells) demonstrated less severe sepsis in the cecal ligation and puncture (CLP) model (with antibiotics), as indicated by survival and other parameters compared with sepsis in the littermate control. The mgmt null protective effect was lost in CLP mice without antibiotics, highlighting the importance of microbial control during sepsis immune modulation. However, an MGMT inhibitor in CLP with antibiotics in WT mice attenuated serum cytokines but not mortality, requiring further studies. In conclusion, an absence of mgmt in macrophages resulted in less severe CLP sepsis, implying a possible influence of guanine DNA methylation and repair in macrophages during sepsis.

Less Severe Lipopolysaccharide-Induced Inflammation in Conditional mgmt-Deleted Mice with LysM-Cre System: The Loss of DNA Repair in Macrophages.

Despite the known influence of DNA methylation from lipopolysaccharide (LPS) activation, data on the O6-methylguanine-DNA methyltransferase (MGMT, a DNA suicide repair enzyme) in macrophages is still lacking. The transcriptomic profiling of epigenetic enzymes from wild-type macrophages after single and double LPS stimulation, representing acute inflammation and LPS tolerance, respectively, was performed. Small interfering RNA (siRNA) silencing of mgmt in the macrophage cell line (RAW264.7) and mgmt null (mgmtflox/flox; LysM-Crecre/-) macrophages demonstrated lower secretion of TNF-α and IL-6 and lower expression of pro-inflammatory genes (iNOS and IL-1ß) compared with the control. Macrophage injury after a single LPS dose and LPS tolerance was demonstrated by reduced cell viability and increased oxidative stress (dihydroethidium) compared with the activated macrophages from littermate control mice (mgmtflox/flox; LysM-Cre-/-). Additionally, a single LPS dose and LPS tolerance also caused mitochondrial toxicity, as indicated by reduced maximal respiratory capacity (extracellular flux analysis) in the macrophages of both mgmt null and control mice. However, LPS upregulated mgmt only in LPS-tolerant macrophages but not after the single LPS stimulation. In mice, the mgmt null group demonstrated lower serum TNF-α, IL-6, and IL-10 than control mice after either single or double LPS stimulation. Suppressed cytokine production resulting from an absence of mgmt in macrophages caused less severe LPS-induced inflammation but might worsen LPS tolerance.

Gut Barrier Damage and Gut Translocation of Pathogen Molecules in Lupus, an Impact of Innate Immunity (Macrophages and Neutrophils) in Autoimmune Disease.

The gut barrier is a single cell layer that separates gut micro-organisms from the host, and gut permeability defects result in the translocation of microbial molecules from the gut into the blood. Despite the silent clinical manifestation, gut translocation of microbial molecules can induce systemic inflammation that might be an endogenous exacerbating factor of systemic lupus erythematosus. In contrast, circulatory immune-complex deposition and the effect of medications on the gut, an organ with an extremely large surface area, of patients with active lupus might cause gut translocation of microbial molecules, which worsens lupus severity. Likewise, the imbalance of gut microbiota may initiate lupus and/or interfere with gut integrity which results in microbial translocation and lupus exacerbation. Moreover, immune hyper-responsiveness of innate immune cells (macrophages and neutrophils) is demonstrated in a lupus model from the loss of inhibitory Fc gamma receptor IIb (FcgRIIb), which induces prominent responses through the cross-link between activating-FcgRs and innate immune receptors. The immune hyper-responsiveness can cause cell death, especially apoptosis and neutrophil extracellular traps (NETosis), which possibly exacerbates lupus, partly through the enhanced exposure of the self-antigens. Leaky gut monitoring and treatments (such as probiotics) might be beneficial in lupus. Here, we discuss the current information on leaky gut in lupus.

Cell-free DNA as diagnostic and prognostic biomarkers for adult sepsis: a systematic review and meta-analysis.

Although cell-free DNA (cfDNA) is an emerging sepsis biomarker, the use of cfDNA, especially as diagnostic and prognostic indicators, has surprisingly not been systemically analyzed. Data of adult patients with sepsis that conducted cfDNA measurement within 24 h of the admission was collected from PubMed, ScienceDirect, Scopus, and Cochrane Library until October 2022. The Quality in Prognosis Studies (QUIPS) and Quality Assessment in Diagnostic Studies-2 (QUADAS-2) tools were used to reduce the risk of biased assessment. The mean difference (MD) of cfDNA concentration and the standardized mean difference (SMD) between populations was calculated using Review Manager (RevMan) version 5.4.1 package software. Pooled analysis from 18 included studies demonstrated increased serum cfDNA levels in sepsis when compared with healthy control (SMD = 1.02; 95% confidence interval (CI) 0.46-1.57) or non-sepsis patients in the intensive care unit (ICU) (SMD = 1.03; 95% CI 0.65-1.40), respectively. Meanwhile, a slight decrease in the statistical value was observed when compared with non-sepsis ICU patients with SIRS (SMD = 0.74; 95% 0.41-1.06). The lower cfDNA levels were also observed in sepsis survivors compared to the non-survivors (SMD at 1.43; 95%CI 0.69-2.17) with the pooled area under the receiver operating characteristic curve (AUC) of 0.76 (95% CI 0.64-0.87) for the mortality prediction. Levels of cfDNA showed a pooled sensitivity of 0.81 (95% CI 0.75-0.86) and specificity of 0.72 (95% CI 0.65-0.78) with pooled diagnostic odd ratio (DOR) at 25.03 (95% CI 5.48-114.43) for the identification of sepsis in critically ill conditions. The cfDNA levels were significantly higher in patients with sepsis and being a helpful indicator for the critically ill conditions of sepsis. Nevertheless, results of the test must be interpreted carefully with the context of all clinical situations.

High-Dose Intravenous Ascorbate in Sepsis, a Pro-Oxidant Enhanced Microbicidal Activity and the Effect on Neutrophil Functions.

Vitamin C (ascorbic acid), a water-soluble essential vitamin, is well-known as an antioxidant and an essential substrate for several neutrophil functions. Because of (i) the importance of neutrophils in microbial control and (ii) the relatively low vitamin C level in neutrophils and in plasma during stress, vitamin C has been studied in sepsis (a life-threatening organ dysfunction from severe infection). Surprisingly, the supraphysiologic blood level of vitamin C (higher than 5 mM) after the high-dose intravenous vitamin C (HDIVC) for 4 days possibly induces the pro-oxidant effect in the extracellular space. As such, HDIVC demonstrates beneficial effects in sepsis which might be due to the impacts on an enhanced microbicidal activity through the improved activity indirectly via enhanced neutrophil functions and directly from the extracellular pro-oxidant effect on the organismal membrane. The concentration-related vitamin C properties are also observed in the neutrophil extracellular traps (NETs) formation as ascorbate inhibits NETs at 1 mM (or less) but facilitates NETs at 5 mM (or higher) concentration. The longer duration of HDIVC administration might be harmful in sepsis because NETs and pro-oxidants are partly responsible for sepsis-induced injuries, despite the possible microbicidal benefit. Despite the negative results in several randomized control trials, the short course HDIVC might be interesting to use in some selected groups, such as against anti-biotic resistant organisms. More studies on the proper use of vitamin C, a low-cost and widely available drug, in sepsis are warranted.

Obesity Exacerbates Lupus Activity in Fc Gamma Receptor IIb Deficient Lupus Mice Partly through Saturated Fatty Acid-Induced Gut Barrier Defect and Systemic Inflammation.

The prevalence of obesity is increasing, and the coexistence of obesity and systemic lupus erythematosus (lupus) is possible. A high-fat diet (HFD) was orally administered for 6 months in female 8-week-old Fc gamma receptor IIb deficient (FcgRIIb-/-) lupus or age and gender-matched wild-type (WT) mice. Lupus nephritis (anti-dsDNA, proteinuria, and increased creatinine), gut barrier defect (fluorescein isothiocyanate dextran), serum lipopolysaccharide (LPS), serum interleukin (IL)-6, liver injury (alanine transaminase), organ fibrosis (liver and kidney pathology), spleen apoptosis (activated caspase 3), and aorta thickness (but not weight gain and lipid profiles) were more prominent in HFD-administered FcgRIIb-/- mice than the obese WT, without injury in regular diet-administered mice (both FcgRIIb-/- and WT). In parallel, combined palmitic acid (PA; a saturated fatty acid) with LPS (PA + LPS) induced higher tumor necrotic factor-α, IL-6, and IL-10 in the supernatant, inflammatory genes (inducible nitric oxide synthase and IL-1ß), reactive oxygen species (dihydroethidium), and glycolysis with reduced mitochondrial activity (extracellular flux analysis) when compared with the activation by each molecule alone in both FcgRIIb-/- and WT macrophages. However, the alterations of these parameters were more prominent in PA + LPS-administered FcgRIIb-/- than in the WT cells. In conclusion, obesity accelerated inflammation in FcgRIIb-/- mice, partly due to the more potent responses from the loss of inhibitory FcgRIIb against PA + LPS with obesity-induced gut barrier defect.

A Synergy Between Endotoxin and (1→3)-Beta-D-Glucan Enhanced Neutrophil Extracellular Traps in Candida Administered Dextran Sulfate Solution Induced Colitis in FcGRIIB-/- Lupus Mice, an Impact of Intestinal Fungi in Lupus.

INTRODUCTION: The translocation of organismal molecules from gut into blood circulation might worsen the disease severity of lupus through the induction of neutrophil extracellular traps (NETs). METHODS: An impact of lipopolysaccharide (LPS) and (1→3)-ß-D-glucan (BG), components of gut bacteria and fungi, respectively, on NETs formation, was explored in lupus models, Fc gamma receptor IIB deficiency (FcGRIIB-/-) and Pristane injection, using Candida-administered dextran sulfate solution induced colitis (Candida-DSS) model. RESULTS: Severity of Candida-DSS in FcGRIIB-/- mice was more prominent than wild-type (WT) and Pristane mice as indicated by (i) colonic NETs using immunofluorescence of Ly6G, myeloperoxidase (MPO) and neutrophil elastase (NE) together with expression of PAD4 and IL-1ß, (ii) colonic immunoglobulin (Ig) deposition (immunofluorescence), (iii) gut-leakage by FITC-dextran assay, endotoxemia and serum BG, (iv) systemic inflammation (neutrophilia, serum cytokines, serum dsDNA and anti-dsDNA) and (v) renal injury (proteinuria, glomerular NETs and Ig deposition). DISCUSSION: The formation of NETs in Candida-DSS mice was more severe than non-Candida-DSS mice and NETs in Candida-DSS were more profound in FcGRIIB-/- mice than Pristane mice. Prominent NETs in Candida-DSS FcGRIIB-/- mice might be due to the profound responses against LPS+BG in FcGRIIB-/- neutrophils compared with WT cells. These data implied an impact of the inhibitory FcGRIIB in NETs formation and an influence of gut fungi in lupus exacerbation. Hence, gut fungi in a DSS-induced gut-leakage lupus model enhanced colonic NETs that facilitated gut translocation of organismal molecules and synergistically exacerbated lupus activity.

BAM15, a Mitochondrial Uncoupling Agent, Attenuates Inflammation in the LPS Injection Mouse Model: An Adjunctive Anti-Inflammation on Macrophages and Hepatocytes.

Controlof immune responses through the immunometabolism interference is interesting for sepsis treatment. Then, expression of immunometabolism-associated genes and BAM15, a mitochondrial uncoupling agent, was explored in a proinflammatory model using lipopolysaccharide (LPS) injection. Accordingly, the decreased expression of mitochondrial uncoupling proteins was demonstrated by transcriptomic analysis on metabolism-associated genes in macrophages (RAW246.7) and by polymerase chain reaction in LPS-stimulated RAW246.7 and hepatocytes (Hepa 1-6). Pretreatment with BAM15 at 24 h prior to LPS in macrophages attenuated supernatant inflammatory cytokines (IL-6, TNF-α, and IL-10), downregulated genes of proinflammatory M1 polarization (iNOS and IL-1ß), upregulated anti-inflammatory M2 polarization (Arg1 and FIZZ), and decreased cell energy status (extracellular flux analysis and ATP production). Likewise, BAM15 decreased expression of proinflammatory genes (IL-6, TNF-α, IL-10, and iNOS) and reduced cell energy in hepatocytes. In LPS-administered mice, BAM15 attenuated serum cytokines, organ injury (liver enzymes and serum creatinine), and tissue cytokines (livers and kidneys), in part, through the enhanced phosphorylated αAMPK, a sensor of ATP depletion with anti-inflammatory property, in the liver, and reduced inflammatory monocytes/macrophages (Ly6C +ve, CD11b +ve) in the liver as detected by Western blot and flow cytometry, respectively. In conclusion, a proof of concept for inflammation attenuation of BAM15 through metabolic interference-induced anti-inflammation on macrophages and hepatocytes was demonstrated as a new strategy of anti-inflammation in sepsis.

Pathogen-Associated Molecules from Gut Translocation Enhance Severity of Cecal Ligation and Puncture Sepsis in Iron-Overload ß-Thalassemia Mice.

INTRODUCTION: Systemic inflammation induced by gut translocation of lipopolysaccharide (LPS), a major component of Gram-negative bacteria, in thalassemia with iron-overload worsens sepsis. However, the impact of (1→3)-ß-D-glucan (BG), a major fungal molecule, in iron-overload thalassemia is still unclear. Hence, the influence of BG was explored in 1) iron-overload mice with sepsis induced by cecal ligation and puncture (CLP) surgery; and 2) in bone marrow-derived macrophages (BMMs). METHODS: The heterozygous ß-globin-deficient mice, Hbbth3/+ mice, were used as representative thalassemia (TH) mice. Iron overload was generated by 6 months of oral iron administration before CLP surgery- induced sepsis in TH mice and wild-type (WT) mice. Additionally, BMMs from both mouse strains were used to explore the impact of BG. RESULTS: Without sepsis, iron-overload TH mice demonstrated more severe intestinal mucosal injury (gut leakage) with higher LPS and BG in serum, from gut translocation, when compared with WT mice. With CLP in iron-overload mice, sepsis severity in TH mice was more severe than WT as determined by survival analysis, organ injury (kidney and liver), bacteremia, endotoxemia, gut leakage (FITC-dextran) and serum BG. Activation by LPS plus BG (LPS+BG) in BMMs and in peripheral blood-derived neutrophils (both WT and TH cells) demonstrated more prominent cytokine production when compared with LPS activation alone. In parallel, LPS+BG also prominently induced genes expression of M1 macrophage polarization (iNOS, TNF-α and IL-1ß) in both WT and TH cells in comparison with LPS activation alone. In addition, LPS+BG activated macrophage cytokine production was enhanced by a high dose of ferric ion (800 mM), more predominantly in TH macrophages compared with WT cells. Moreover, LPS+BG induced higher glycolysis activity with similar respiratory capacity in RAW264.7 (a macrophage cell line) compared with LPS activation alone. These data support an additive pro-inflammatory effect of BG upon LPS. CONCLUSION: The enhanced-severity of sepsis in iron-overload TH mice was due to 1) increased LPS and BG in serum from iron-induced gut-mucosal injury; and 2) the pro-inflammatory amplification by ferric ion on LPS+BG activation.

Defective Neutrophil Function in Patients with Sepsis Is Mostly Restored by ex vivo Ascorbate Incubation.

BACKGROUND: Neutrophil function is essential for effective defence against bacterial infections but is defective in patients with sepsis. Ascorbate or vitamin C, which is low in the plasma of patients with sepsis, is stored inside human neutrophils and is essential for their normal function. OBJECTIVE: This study aimed to determine if ascorbate treatment ex vivo improved neutrophil function in patients with sepsis. PATIENTS AND METHODS: Human blood neutrophils were isolated from 20 patients with sepsis and 20 healthy age-matched controls. Neutrophils were incubated with or without ascorbate (1, 5, 10, 20 and 40 mM) for periods up to 2h. Chemotaxis was evaluated using a chemotactic chamber in response to the chemoattractant, fMLP. Phagocytosis (uptake of pHrodo red stained S. aureus) and apoptosis (annexin-V/propidium iodide staining) were measured by flow cytometry. Neutrophil extracellular trap (NET) formation was detected and quantified using DAPI, anti-myeloperoxidase and anti-neutrophil elastase immuno-fluorescence staining. Quantifluor detected the amount of dsDNA in NET supernatants, while quantitative PCR identified changes in expression of PADI4 gene. RESULTS: Chemotactic and phagocytic activities were decreased in patients with sepsis but increased after treatment with the high concentrations of ascorbate. Apoptosis was increased in the sepsis patients but not altered by ascorbate treatment. Spontaneous NET formation was observed in patients with sepsis. A quantity of 1mM ascorbate decreased spontaneous NETosis to that of normal, healthy neutrophils, while high concentrations of ascorbate (>10mM) further promoted NET formation. CONCLUSION: Dysregulated neutrophil function was observed in patients with sepsis which could contribute to disease pathology and outcomes. Exposure to ascorbate could reverse some of these changes in function. These novel discoveries raise the possibility that ascorbate treatment could be used as an adjunctive therapy that could result in improved neutrophil function during sepsis.