Inflammatory Cytokine Response Research Articles

Frequency of IRF5+ dendritic cells is associated with the TLR7-induced inflammatory cytokine response in SARS-CoV-2 infection.

The SARS-CoV-2 infection leads to enhanced inflammation driven by innate immune responses. Upon TLR7 stimulation, dendritic cells (DC) mediate the production of inflammatory cytokines, and in particular of type I interferons (IFN). Especially in DCs, IRF5 is a key transcription factor that regulates pathogen-induced immune responses via activation of the MyD88-dependent TLR signaling pathway. In the current study, the frequencies of IRF5+ DCs and the association with innate cytokine responses in SARS-CoV-2 infected individuals with different disease courses were investigated. In addition to a decreased number of mDC and pDC subsets, we could show reduced relative IRF5+ frequencies in mDCs of SARS-CoV-2 infected individuals compared with healthy donors. Functionally, mDCs of COVID-19 patients produced lower levels of IL-6 in response to in vitro TLR7 stimulation. IRF5+ mDCs more frequently produced IL-6 and TNF-α compared to their IRF5- counterparts upon TLR7 ligation. The correlation of IRF5+ mDCs with the frequencies of IL-6 and TNF-α producing mDCs were indicators for a role of IRF5 in the regulation of cytokine responses in mDCs. In conclusion, our data provide further insights into the underlying mechanisms of TLR7-dependent immune dysfunction and identify IRF5 as a potential immunomodulatory target in SARS-CoV-2 infection.

Therapeutic Targeting of Inflammation and Virus Simultaneously Ameliorates Influenza Pneumonia and Protects from Morbidity and Mortality.

Influenza pneumonia is a severe complication caused by inflammation of the lungs following infection with seasonal and pandemic strains of influenza A virus (IAV), that can result in lung pathology, respiratory failure, and death. There is currently no treatment for severe disease and pneumonia caused by IAV. Antivirals are available but are only effective if treatment is initiated within 48 h of onset of symptoms. Influenza complications and mortality are often associated with high viral load and an excessive lung inflammatory cytokine response. Therefore, we simultaneously targeted the virus and inflammation. We used the antiviral oseltamivir and the anti-inflammatory drug etanercept to dampen TNF signaling after the onset of clinical signs to treat pneumonia in a mouse model of respiratory IAV infection. The combined treatment down-regulated the inflammatory cytokines TNF, IL-1β, IL-6, and IL-12p40, and the chemokines CCL2, CCL5, and CXCL10. Consequently, combined treatment with oseltamivir and a signal transducer and activator of transcription 3 (STAT3) inhibitor effectively reduced clinical disease and lung pathology. Combined treatment using etanercept or STAT3 inhibitor and oseltamivir dampened an overlapping set of cytokines. Thus, combined therapy targeting a specific cytokine or cytokine signaling pathway and an antiviral drug provide an effective treatment strategy for ameliorating IAV pneumonia. This approach might apply to treating pneumonia caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Myeloid miR-155 plays a limited role in antibacterial defense during Klebsiella-derived pneumosepsis and is dispensable for lipopolysaccharide- or Klebsiella-induced inflammation in mice.

MicroRNA-155 (miR-155) plays a crucial role in regulating host inflammatory responses during bacterial infection. Previous studies have shown that constitutive miR-155 deficiency alleviates inflammation while having varying effects in different bacterial infection models. However, whether miR-155 in myeloid cells is involved in the regulation of inflammatory and antibacterial responses is largely elusive. Mice with myeloid cell specific miR-155 deficiency were generated to study the in vitro response of bone marrow-derived macrophages (BMDMs), alveolar macrophages (AMs) and peritoneal macrophages (PMs) to lipopolysaccharide (LPS), and the in vivo response after intranasal or intraperitoneal challenge with LPS or infection with Klebsiella (K.) pneumoniae via the airways. MiR-155 deficient macrophages released less inflammatory cytokines than control macrophages upon stimulation with LPS in vitro. However, the in vivo inflammatory cytokine response to LPS or K. pneumoniae was not affected by myeloid miR-155 deficiency. Moreover, bacterial outgrowth in the lungs was not altered in myeloid miR-155 deficient mice, but Klebsiella loads in the liver of these mice were significantly higher than in control mice. These data argue against a major role for myeloid miR-155 in host inflammatory responses during LPS-induced inflammation and K. pneumoniae-induced pneumosepsis but suggest that myeloid miR-155 contributes to host defense against Klebsiella infection in the liver.

DUOX2 regulates secreted factors in virus-infected respiratory epithelial cells that contribute to neutrophil attraction and activation.

The first line of defense against respiratory viruses relies on the antiviral and proinflammatory cytokine response initiated in infected respiratory epithelial cells. The cytokine response not only restricts virus replication and spreading, but also orchestrates the subsequent immune response. The epithelial Dual Oxidase 2 (DUOX2) has recently emerged as a regulator of the interferon antiviral response. Here, we investigated the role of DUOX2 in the inflammatory cytokine response using a model of A549 cells deficient in DUOX2 generated using Crispr-Cas9 and infected by Sendai virus. We found that the absence of DUOX2 selectively reduced the induction of a restricted panel of 14 cytokines and chemokines secreted in response to Sendai virus by 20 to 89%. The secreted factors produced by epithelial cells upon virus infection promoted the migration, adhesion, and degranulation of primary human neutrophils, in part through the DUOX2-dependent secretion of TNF and chemokines. In contrast, DUOX2 expression did not impact neutrophil viability or NETosis, thereby highlighting a selective impact of DUOX2 in neutrophil functions. Overall, this study unveils previously unrecognized roles of epithelial DUOX2 in the epithelial-immune cells crosstalk during respiratory virus infection.

Meningitis-associated pneumococcal serotype 8, ST 53, strain is hypervirulent in a rat model and has non-haemolytic pneumolysin which can be attenuated by liposomes.

Streptococcus pneumoniae bacteria cause life-threatening invasive pneumococcal disease (IPD), including meningitis. Pneumococci are classified into serotypes, determined by differences in capsular polysaccharide and both serotype and pneumolysin toxin are associated with disease severity. Strains of serotype 8, ST 53, are increasing in prevalence in IPD in several countries. Here we tested the virulence of such an isolate in a rat model of meningitis in comparison with a serotype 15B and a serotype 14 isolate. All three were isolated from meningitis patients in South Africa in 2019, where serotype 8 is currently the most common serotype in IPD. Only the serotype 8 isolate was hypervirulent causing brain injury and a high mortality rate. It induced a greater inflammatory cytokine response than either the serotype 15B or 14 strain in the rat model and from primary mixed-glia cells isolated from mouse brains. It had the thickest capsule of the three strains and produced non-haemolytic pneumolysin. Pneumolysin-sequestering liposomes reduced the neuroinflammatory cytokine response in vitro indicating that liposomes have the potential to be an effective adjuvant therapy even for hypervirulent pneumococcal strains with non-haemolytic pneumolysin.

The Correlation Between Cardiac Oxidative Stress and Inflammatory Cytokine Response Following Myocardial Infarction.

Our study was conducted to investigate the potential correlation between cardiac oxidative stress and inflammatory cytokine response following myocardial infarction. A total of 120 patients harboring acute myocardial infarction who underwent percutaneous coronary intervention (PCI) at our hospital were included. Their general clinical data were analyzed, and comparisons were made regarding the levels of inflammatory factors, oxidative stress markers, heart pump function, and cardiac function. The correlation between cardiac oxidative stress and inflammatory cytokine response was assessed using Pearson's linear correlation. Following treatment, significant reductions were seen in the serum levels of cortisol, thyroid-stimulating hormone (TSH), B-type natriuretic peptide (BNP), C-reactive protein (CRP), signal transducer and activator of transcription 3 (STAT3), interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor (TNF), and catalase (CAT) compared to pre-treatment levels. Conversely, the levels of growth hormone (GH), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), total antioxidant capacity (T-AOC), cardiac output (CO), and cardiac index (CI) were significantly elevated. Serum cortisol (r = 0.481, P = .001), BNP (r = 0.437, P = .001), CRP (r = 0.542, P = .001), STAT3 (r = 0.835, P = .001), TSH (P = .001), IL-8 (r = 0.867, P = .001), TNF-α (r = 0.439, P = .001), and cardiac oxidative stress demonstrated significantly positive correlations (P < .05). Additionally, a significant negative correlation was found between GH (r = -0.654, P = .001) and immune balance (P < .05). This study evaluated the severity of myocardial infarction using indicators such as CO and CI. This study found a significant correlation between cardiac oxidative stress and inflammatory cytokines after myocardial infarction, suggesting their potential as predictors of myocardial infarction severity.

1,25-Dihydroxy Vitamin D3 Facilitates the M2 Polarization and β-Amyloid Uptake by Human Microglia in a TREM2-Dependent Manner.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by dementia as the primary clinical symptom. The production and accumulation of aggregated β-amyloid (Aβ) in patient brain tissues is one of the hallmarks of AD pathogenesis. Microglia, brain-resident macrophages, produce inflammatory cytokines in response to Aβ oligomers or fibrils exacerbating Aβ pathology in AD. HMO6 cells were treated with Aβ42 in the presence or absence of 1,25-dihydroxy vitamin D3 (1,25(OH)2D3) to determine its potential immunomodulatory effects, and the expression of pro-/anti-inflammatory cytokines, M1/M2-associated markers, Toll-like receptors (TLRs), and triggering receptor expressed on myeloid cells 2 (TREM2) was examined. 1,25(OH)2D3 was found to suppress Aβ-induced expression of proinflammatory cytokines (TNF-α, IL-1β, and IL-6), M1 markers (CD86 and iNOS), and TLR2/4, whilst increasing the expression of anti-inflammatory cytokines (IL-4, IL-10, and CCL17) and M2 markers (CD206 and Arg-1). Furthermore, 1,25(OH)2D3 promoted TREM2 expression and Aβ uptake by HMO6 cells, and the enhancement of Aβ uptake and M2 polarization was revealed to be TREM2-dependent. The findings of this study suggest that 1,25(OH)2D3 facilitates M2 polarization and Aβ uptake in a TREM2-dependent manner.

A comparative study of pectic polysaccharides from fresh and dried Dendrobium officinale based on their structural properties and hepatoprotection in alcoholic liver damaged mice.

In this study, two pectic polysaccharides from fresh and dried Dendrobium officinale, namely FDP and DDP, were obtained by sour-water extraction, ethanol precipitation and further purification with DEAE cellulose-52 and Sephadex G-100 column chromatography. FDP/DDP had eight similar glycosidic linkages including 1,4-linked-GlcAp, 1,4- and 1,3,4-linked-GalAp, 1,3,4- and T-linked-Glcp, 1,6- and T-linked-Galp, T-linked-Galp and T-linked-Xylp. Besides, FDP was marked by 1,6-, 1,2,6-linked-Manp and 1,2,4-, 1,2-linked-Rhap, and DDP consisted of unique 1,6-linked-GlcAp and 1,3,6-Manp. FDP with a molecular weight of 14.8 kDa generally showed stronger scavenging capacity against DPPH, ABTS and hydroxyl radicals than DDP (p < 0.05). Pretreatment with FDP/DDP alleviated the alcohol-induced liver injury in mice, and their serum aminotransferase and triglyceride levels were 10.3%-57.8% lower than those of the model group (MG). Meanwhile, the FDP/DDP-M and FDP/DDP-H groups (200 and 300 mg kg-1) displayed a remarkable increase in antioxidant enzyme activities and significant reduction in inflammatory cytokine levels in comparison with the MG. Further analysis revealed that FDP-treated mice generally exhibited lower transaminase levels and inflammatory cytokine expression as well as higher antioxidant enzyme activities than DDP-treated ones. The FDP-H group showed significant restoration, which was slightly less than or almost comparable to that of the bifendate-fed positive control. The above results indicate that D. officinale pectin can attenuate oxidative stress and inflammatory cytokine response, and ultimately ameliorate liver injury, and "fresh" pectin with specific structural characteristics is expected to be more promising as hepatoprotective food.

The acute leukocyte and cytokine response of older adults to resistance exercise in normobaric hypoxia.

Ageing causes a decline in leukocyte function and blunted leukocyte responses to resistance exercise. Systemic hypoxia exposure augments the leukocyte response to resistance exercise in young adults, yet this response remains uncharacterised in older adults. This study characterised the effects of normobaric hypoxia on the acute leukocyte and inflammatory cytokine responses to resistance exercise in older adults. We recruited 20 adults aged 60-70 years to perform an acute bout of resistance exercise in normobaric hypoxia (FiO2 14.4%; n = 10) or normoxia (FiO2 20.93%; n = 10). Participants completed 4 × 10 repetitions of lower and upper body exercises at 70% of their predicted 1-repetition maximum. Venous blood was sampled before and up to 24 hours post-exercise to quantify neutrophils, lymphocytes, monocytes, eosinophils, basophils and cytokines (IL-1β, IL-4, IL-6, IL-8, IL-10, TNFα). Flow cytometry was used to classify lymphocytes as T (CD4+ helper and CD8+ cytotoxic), B and NK cells, in addition to the expression of the senescence marker CD45RA on T cells. The hypoxic group showed a larger lymphocyte response over the 24 hours post-exercise compared to the normoxic group (p = 0.035). Specifically, there were greater concentrations of CD4+ T helper cells following hypoxic exercise compared to normoxia (p = 0.046). There was also a greater proportion of CD45RA+ CD4+ T helper cells, suggesting that the cells were more senescent (p = 0.044). Hypoxia did not impact any other leukocyte population or cytokine following exercise. Normobaric hypoxia increases the lymphocyte response to an acute bout of resistance exercise in older adults.

Exogenous antibiotic resistance gene contributes to intestinal inflammation by modulating the gut microbiome and inflammatory cytokine responses in mouse

ABSTRACT Dysregulation of the gut microbiota by environmental factors is associated with a variety of autoimmune and immune-mediated diseases. In addition, naturally-occurring extracellular antibiotic resistance genes (eARGs) might directly enter the gut via the food chain. However, following gut microbiota exposure to eARGs, the ecological processes shaping the microbiota community assembly, as well as the interplay between the microbiota composition, metabolic function, and the immune responses, are not well understood. Increasing focus on the One Health approach has led to an urgent need to investigate the direct health damage caused by eARGs. Herein, we reveal the significant influence of eARGs on microbiota communities, strongly driven by stochastic processes. How eARGs-stimulate variations in the composition and metabolomic function of the gut microbiota led to cytokine responses in mice of different age and sex were investigated. The results revealed that cytokines were significantly associated with immunomodulatory microbes, metabolites, and ARGs biomarkers. Cytokine production was associated with specific metabolic pathways (arachidonic acid and tryptophan metabolic pathways), as confirmed by ex vivo cytokine responses and recovery experiments in vivo. Furthermore, the gut microbial profile could be applied to accurately predict the degree of intestinal inflammation ascribed to the eARGs (area under the curve = 0.9616). The present study provided a comprehensive understanding of the influence of an eARGs on immune responses and intestinal barrier damage, shedding light on the interplay between eARGs, microbial, metabolites, and the gut antibiotic resistome in modulating the human immune system.

Dickkopf1 fuels inflammatory cytokine responses

Many human diseases, including cancer, share an inflammatory component but the molecular underpinnings remain incompletely understood. We report that physiological and pathological Dickkopf1 (DKK1) activity fuels inflammatory cytokine responses in cell models, mice and humans. DKK1 maintains the elevated inflammatory tone of cancer cells and is required for mounting cytokine responses following ligation of toll-like and cytokine receptors. DKK1-controlled inflammation derives from cell-autonomous mechanisms, which involve SOCS3-restricted, nuclear RelA (p65) activity. We translate these findings to humans by showing that genetic DKK1 variants are linked to elevated cytokine production across healthy populations. Finally, we find that genetic deletion of DKK1 but not pharmacological neutralization of soluble DKK1 ameliorates inflammation and disease trajectories in a mouse model of endotoxemia. Collectively, our study identifies a cell-autonomous function of DKK1 in the control of the inflammatory response, which is conserved between malignant and non-malignant cells. Additional studies are required to mechanistically dissect cellular DKK1 trafficking and signaling pathways.

SULFORAPHANE ADMINISTRATION AFTER HEMORRHAGIC SHOCK/RESUSCITATION IN MICE REDUCES THE SECRETION OF INFLAMMATORY CYTOKINES AND INCREASES THE IMMUNOCOMPETENCE OF SPLENIC MACROPHAGES.

Objective : The purpose of this study was to investigate the immunomodulatory effects of sulforaphane (SFN), a nuclear factor erythroid 2-related factor (Nrf2) pathway activator, on splenic macrophages' immunocompetence after hemorrhagic shock/resuscitation (HS/R). Methods : Male C57/BL6 wild-type mice (n = 6 per group) were subjected to either pressure-controlled HS (MAP, 35-45 mm Hg) or a sham procedure surgery (without HS). After 90 minutes of HS, fluid resuscitation with withdrawn blood and 0.9% NaCl was performed. Sulforaphane (50 mg/kg of body weight) was applied intraperitoneally immediately after the resuscitation phase as well as 24 and 48 h thereafter, depending on group allocation. The mice were killed at 6, 24, and 72 h after resuscitation. After killing, spleens were harvested to perform Nrf2 immunofluorescence histology. Splenic macrophages were isolated and cultured to measure cytokine secretion in the cell culture supernatant. Furthermore, macrophages isolated after 24-hour resuscitation were treated with 100 ng/mL of bacterial LPS to measure immunocompetence. Matrix-assisted laser desorption/ionization mass spectrometry imaging was performed to verify the distribution of SFN in the spleen after intraperitoneal injection. Results : We showed that administered SFN reached the spleen within the first hour after administration. Furthermore, we identified that SFN increased splenic Nrf2 activation and decreased cytokine expression in splenic macrophages after HS/R. In addition, we showed that SFN exhibited splenic anti-inflammatory properties of macrophages in vitro (IL-6/IL-10-ratio of the HS/R group: 51.79 ± 9.99 [at 6 h] and 15.70 ± 3.35 [at 24 h] vs. HS/R + SFN group: 20.54 ± 5.35 [at 6 h] and 8.60 ± 2.37 [at 24 h], P < 0.05). Furthermore, SFN improved in vitro splenic macrophage immunocompetence after HS/R, as evidenced by the increased secretion of inflammatory cytokines in response to LPS stimulation in vitro . Conclusions : Our study shows that SFN can reduce inflammatory cytokines secreted by splenic macrophages after HS/R and increase their immunocompetence toward a more anti-inflammatory profile.

A Comparative Study of Two Models of Intraluminal Filament Middle Cerebral Artery Occlusion in Rats: Long-Lasting Accumulation of Corticosterone and Interleukins in the Hippocampus and Frontal Cortex in Koizumi Model.

Recently, we have shown the differences in the early response of corticosterone and inflammatory cytokines in the hippocampus and frontal cortex (FC) of rats with middle cerebral artery occlusion (MCAO), according to the methods of Longa et al. (LM) and Koizumi et al. (KM) which were used as alternatives in preclinical studies to induce stroke in rodents. In the present study, corticosterone and proinflammatory cytokines were assessed 3 months after MCAO. The most relevant changes detected during the first days after MCAO became even more obvious after 3 months. In particular, the MCAO-KM (but not the MCAO-LM) group showed significant accumulation of corticosterone and IL1β in both the ipsilateral and contralateral hippocampus and FC. An accumulation of TNFα was detected in the ipsilateral hippocampus and FC in the MCAO-KM group. Thus, unlike the MCAO-LM, the MCAO-KM may predispose the hippocampus and FC of rats to long-lasting bilateral corticosterone-dependent distant neuroinflammatory damage. Unexpectedly, only the MCAO-LM rats demonstrated some memory deficit in a one-trial step-through passive avoidance test. The differences between the two MCAO models, particularly associated with the long-lasting increase in glucocorticoid and proinflammatory cytokine accumulation in the limbic structures in the MCAO-KM, should be considered in the planning of preclinical experiments, and the interpretation and translation of received results.

Immunity Cell Responses to RSV and the Role of Antiviral Inhibitors: A Systematic Review.

Antigen-presenting cells recognize respiratory syncytial virus antigens, and produce cytokines and chemokines that act on immune cells. Dendritic cells play the main role in inflammatory cytokine responses. Similarly, alveolar macrophages produce IFN-β, IFN-α, TNF-α, IL-6, CXCL10, and CCL3, while alternatively activated macrophages differentiate at the late phase, and require IL-13 or IL-4 cytokines. Furthermore, activated NKT cells secrete IL-13 and IL-4 that cause lung epithelial, endothelial and fibroblasts to secrete eotaxin that enhances the recruitment of eosinophil to the lung. CD8+ and CD4+T cells infection by the virus decreases the IFN-γ and IL-2 production. Despite this, both are involved in terminating virus replication. CD8+T cells produce a larger amount of IFN-γ than CD4+T cells, and CD8+T cells activated under type 2 conditions produce IL-4, down regulating CD8 expression, granzyme and IFN-γ production. Antiviral inhibitors inhibit biological functions of viral proteins. Some of them directly target the virus replication machinery and are effective at later stages of infection; while others inhibit F protein dependent fusion and syncytium formation. TMC353121 reduces inflammatory cytokines, TNF-α, IL-6, and IL-1β and chemokines, KC, IP-10, MCP and MIP1-α. EDP-938 inhibits viral nucleoprotein (N), while GRP-156784 blocks the activity of respiratory syncytial virus ribonucleic acid (RNA) polymerase. PC786 inhibits non-structural protein 1 (NS-1) gene, RANTES transcripts, virus-induced CCL5, IL-6, and mucin increase. In general, it is an immune reaction that is blamed for the disease severity and pathogenesis in respiratory syncytial virus infection. Anti-viral inhibitors not only inhibit viral entry and replication, but also may reduce inflammatory cytokines and chemokines. Many respiratory syncytial virus inhibitors are proposed; however, only palivizumab and ribavirin are approved for prophylaxis and treatment, respectively. Hence, this review is focused on immunity cell responses to respiratory syncytial virus and the role of antiviral inhibitors.

Altered Gastric Microbiota and Inflammatory Cytokine Responses in Patients with Helicobacter pylori-Negative Gastric Cancer.

The role of the gastric mucosal microbiome in Helicobacter pylori-negative gastric cancer (GC) remains unclear. Therefore, we aimed to characterize the microbial alterations and host inflammatory cytokine responses in H. pylori-negative GC. Gastric mucosal samples were obtained from 137 H. pylori-negative patients with GC (n = 45) and controls (chronic gastritis or intestinal metaplasia, n = 92). We performed 16S rRNA gene sequencing (n = 67), a quantitative reverse transcription-polymerase chain reaction to determine the relative mRNA expression levels of TNF (tumor necrosis factor), IL1B (interleukin 1 beta), IL6 (interleukin 6), CXCL8 (C-X-C motif chemokine ligand 8), IL10 (interleukin 10), IL17A (interleukin 17A), TGFB1 (transforming growth factor beta 1) (n = 113), and the correlation analysis between sequencing and expression data (n = 47). Gastric mucosal microbiota in patients with GC showed reduced diversity and a significantly different composition compared to that of the controls. Lacticaseibacillus was significantly enriched, while Haemophilus and Campylobacter were depleted in the cancer group compared to the control group. These taxa could distinguish the two groups in a random forest algorithm. Moreover, the combined relative abundance of these taxa, a GC microbiome index, significantly correlated with gastric mucosal IL1B expression, which was elevated in the cancer group. Overall, altered gastric mucosal microbiota was found to be associated with increased mucosal IL1B expression in H. pylori-negative GC.

Functionality of a Peripheral Blood Derived Microglia-like Cell System for Drug Discovery to Treat Sickle Cell Related Chronic Pain

Background: Abnormal microglial activation is emerging as a clinically promising drug target to treat chronic pain in sickle cell disease (SCD). But research involving microglia has been stymied by difficulty in obtaining human CNS microglia and differences between human and animal neurobiology. To address such limitations, we have developed an in vitro culture system to induce human microglia-like cells (MLC) from peripheral blood mononuclear cells. In this study, we further validated the functionality of the cultured peripheral blood derived microglia-like cell (PB-MLC) and evaluate the pathological characteristics captured in the PB-MLC model system, which is an important requirement for disease model systems used in drug discovery. Methods: Human peripheral blood mononuclear cells were cultured with human GM-CSF (10 ng/ml), human IL-34 (100 ng/ml), TGF-β2 (2 ng/mL) and cholesterol (1.5 µg/mL) for 7 days to induce Human peripheral blood derived microglia-like cells (HPB-MLC). HPB-MLC were morphologically analyzed by phase contrast microscopy, phenotyped by immunofluorescence staining with anti-TMEM119, P2RY12, and Iba1 antibodies. To validate that the HPB-MLC culture system recapitulates important pathological features, we compared the microglial morphology, activation states and phagocytic activity of HPB-MLC from individuals with SCD with or without chronic pain, individuals with headache and normal donors without pain: Cells were stimulated with 100 ng/ml LPS for 24 hours, and secretion of TNF-alpha, IL-1beta, and IL-6 was measured by ELISA. The microglial phagocytic function of HPB-MLC was determined by assessing the uptake of fluorescent-labeled Zymosan particles with fluorescent microscopy and imaging flow cytometry. The model system was also validated by comparing cultured PB-MLC to CNS derived microglia cells using Sprague-Dawley rats: Rat brain derived microglia (BDM) and rat peripheral blood monocytes were isolated and then cultured with murine IL-34 (100 ng/ml), murine GM-CSF (10 ng/ml). TGF-β2 (2 ng/mL), and cholesterol (1.5 µg/mL) for 7 days. The cultured cells were morphologically analyzed as above; phenotyped with anti-TMEM119, P2RY12, and Iba1 antibodies. Rat BDM, rat PB-MLC and HPB-MLC were treated with 100 ng/ml LPS with or without minocycline (2.5, 5, 10, 25, 50 μg/mL) for 24 hours; TNF-alpha secretion was measured by ELISA. Results: Rat PB-MLC resemble BDM morphologically and express microglia specific markers. HPB-MLC express microglia specific markers (TMEM119, P2RY12, Iba1), exhibit microglial phagocytic activity. HPB-MLC retain the patient pain phenotype: Cultures from donors without chronic pain show more ramified morphology and lower phagocytic activity than cultures from individuals with chronic pain (Figure 1); HPB-MLC can be activated by LPS, causing microglial deramification and increased the expression of microglial activation marker CD68. HPB-MLC from patients with pain are more easily activated and secrete more inflammatory cytokines in response to LPS; SCD pain and headache samples exhibit different pro-inflammatory cytokine profile. To evaluate the possibility of using the PB-MLC model system to screen compounds to inhibit microglia activation, we tested PB-MLC cells with the microglial inhibitor, minocycline. The compound significantly suppressed the release of proinflammatory cytokine TNF-alpha from LPS-induced activated PB-MLC in a dose-dependent manner (Figure 2). Conclusions: We have developed and validated a microglial-like cell culture system from the peripheral blood of patients with sickle cell disease that retains the patient pain phenotype. HPB-MLC are physiologically functional and exhibit mature microglial features, including LPS-induced activation, microglial specific morphological transition, microglial phagocytic activity, and drug response to a well-studied microglia inhibitor, minocycline. As a cost-effective, efficient, and patient-specific microglia model, HPB-MLC culture system can be applied to identify target compounds that normalize microglia activation, and potentially treat chronic pain. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Rituximab Induces an Inflammatory Cytokine Response with Increased IP-10 (CXCR10) in Patients with Infusion Reactions

Most patients initiating anti-CD20 monoclonal antibody (mAb) therapy for CLL have a disruptive infusion reaction that can cause serious complications and be fatal. A better understanding of the risk factors and etiology of infusion reactions is required to improve preventative measures and management. We report a study to test the hypothesis that IV rituximab induces an inflammatory cytokines response with differences in individual cytokine concentrations correlating with infusion reactions. We designed a clinical trial (NCT03788291) for initial treatment of patients with progressive CLL that provided clinical data and blood specimens from 37 patients treated with 50 mg of IV rituximab infused at 25 mg/h. Blood specimens were collected immediately prior to initiation of rituximab (baseline), one hour later (1h), at the end of the infusion (post-infusion) and at 48 h after initiation of therapy (48h). Infusion reactions (CTCAE grade ≥2 events) were managed by interrupting the infusion and initiating symptomatic treatment. We measured circulating CLL cell counts and CD20 levels, and serum rituximab concentration, complement levels, and cytokine concentrations of IFN-γ, IL-10, IL-18, IL-2, IL-4, IL-6, IL-8, IP-10, MCP-1, MIP-1α, MIP-1β, and TNF-α using standard methods. Twenty-four (65%) patients had 25 infusion reactions (all CTCAE grade 2) at a median of 78 minutes (range 35-128) after initiation of rituximab therapy (median dose 32 mg, range 15-50). All patients completed the 50 mg rituximab infusion (median time 150 minutes, range 119-195). Patient and CLL characteristics including baseline CLL cell counts did not predict the risk of infusion reactions. Median CLL counts at 1h decreased to 14.7% of baseline with no further changes post-infusion and no significant difference between patients with or without infusion reactions (60x109/L vs. 45x109/L, p=0.61). Median CLL cell membrane CD20 levels (x103 molecules/cell) decreased from 4.77 at baseline to 3.10 at 1h (p=0.002) and 1.59 post-infusion (p<0.0001 vs. baseline and 1h) with no significant differences between patients with or without an infusion reaction. The median estimated number of circulating CLL cell membrane CD20 molecules (x1012 molecules/L) decreased from 325.8 at baseline to 34.1 at 1h (p<0.0001) and 14.5 post-infusion (p<0.0001 vs. baseline, p<0.03 vs. 1h) with no significant differences between patients with or without an infusion reaction. There was no correlation between risk of infusion reaction and serum levels of rituximab or serum complement. Thirty-five (95%) patients had a ≥4 fold increase in serum concentration of ≥1 cytokine with resolution of the cytokine response at 48h. All patients with an infusion reaction had significantly higher median post-infusion serum concentrations of IP-10, IL-6 and IL-8. All 17 patients with IP-10 serum concentrations above the limit of detection (40,000 pg/ml) post-infusion had an infusion reaction, and all 24 patients with an infusion reaction had a ≥4 fold increase in serum IP-10 concentrations. Of 13 patients without an infusion reaction, the highest measured serum IP-10 concentration was 22,013 pg/ml and only 3 (23%) had ≥4 fold increases in serum IP-10 concentrations. Slow infusion of a reduced dose of rituximab induced a cytokine response in 95% of patients and therapy disrupting infusion reactions occurred at a comparable rate (65%) to standard rituximab therapy. Infusion reactions were associated with increased levels of IP-10, IL-6 and IL-8. IP-10 levels were increased by ≥4 fold in all patients with an infusion reaction compared to 23% without an infusion reaction. These data suggest that rituximab infusion reactions are caused by inflammatory cytokines produced by cytotoxic effector cells activated by rituximab independent of the pre-treatment amount of circulating CLL cells or CD20 levels, or efficacy of rituximab in reducing the circulating CLL cell count. Infusion reactions occurred after at least 35 minutes of therapy concurrent with a rapid decrease in circulating CLL cells. This observation suggests that cytokine release could be a result of activation of effector cells responsible for CLL cell elimination. Our data provides novel insights into the potential mechanism of infusion reactions and could form the basis of further research to determine methods to control this clinically important and therapy limiting complication.

Secreted Sars-Cov-2 Encoded ORF8 Protein Stimulates Pro-Inflammatory Cytokines and Predicts Severe Clinical Outcome

Introduction: Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can result in a cytokine storm that is associated with poor patient outcomes. Despite extensive research, the specific factor associated with SARS-CoV-2 infection that mediates the inflammatory response remains unidentified. We hypothesized that one or more of these proteins is secreted to the bloodstream to induce a systematic inflammatory cytokine response leading to the cytokine storm in patients. We demonstrate that SARS-CoV-2 encoded Open Reading Frame 8 (ORF8) protein is abundantly secreted as a glycoprotein into culture supernatant in vitro and into the bloodstream in patients with COVID-19. Glycosylated ORF8 stimulates monocytes to produce a set of pro-inflammatory cytokines through an NOD-like receptor family pyrin domain-containing 3 (NLPR3)-mediated inflammasome response, which can be inhibited by select NLRP3 inhibitors. Methods: Following institutional review board approval, serum samples were collected from Mayo Clinic COVID-19 infected patients. Lentiviral constructs expressing SARS-CoV-2 proteins were used to express SARS-CoV-2 proteins in HEK293 cells. Polymerase chain reaction and Luminex multiplex assays were used to detect cytokines induced by COVID-19 proteins. Single-cell RNA sequencing (scRNA-Seq) and flow cytometry were used to identify specific leukocyte populations responsible for cytokine release upon ORF8 stimulation. Immunoblotting, mass spectroscopy, and immunofluorescence microscopy were used to quantify and localize proteins of interest. Results: We transduced HEK293 cells with lentiviral constructs that express each SARS-CoV-2 protein and identified ORF8 as abundantly secreted. We treated human peripheral blood mononuclear cells (PBMCs) from healthy donors in vitro with conditioned media (CM) containing major secreted non-structural proteins (NSP) or secreted ORF8 followed by an assessment of cytokine mRNA expression. We found that none of the secreted NSP proteins induced cytokine expression while ORF8-containing CM (ORF8-CM) induced the mRNA expression of IL1b, IL6, IL8, and CCL2 up to 5-fold in PBMCs from select donors. scRNA-Seq on PBMCs treated with ORF8-CM showed that treatment with ORF8 induced IL1b, IL8, and CCL2 expression in CD14, CD16, CD68, and HLA-DR positive activated monocytes. Flow cytometry analysis of PBMCs stimulated with purified ORF8 showed significant increases of IL1β protein expression only in classical (CD14+/CD16-) and intermediate (CD14+/CD16-) monocyte subsets while no significant changes to cells of other types. scRNA-Seq and Western analyses demonstrated that ORF8 activates NOD-like receptor family pyrin domain-containing 3 (NLPR3) leading to the production of IL1b protein. Furthermore, ORF8-mediated IL1β cytokine induction was abolished in NLPR3 knockdown THP1 cells, demonstrating the NLRP3 dependency of ORF8-mediated cytokine response in human monocytes. We then examined the correlation between blood ORF8 levels and patients’ survival outcome in 25 patients with COVID-19 infection (Figure 1a). After 120 days of follow-up, all seven fatalities were exclusively associated with the ORF8-high group while all patients in ORF8-low group had minimal morbidity and without any events of death (Figure 1b). Importantly, the ORF8-initiated NLPR3-mediated inflammasome pathway can be effectively inhibited by the NLRP3 inhibitor MCC950 at a nanomolar dose range, demonstrating that targeting ORF8/NLRP3 is a promising strategy for treating patients with symptomatic COVID-19. Conclusion: We demonstrate that the virus-encoded ORF8 protein is abundantly secreted as a glycoprotein in vitro and in patients with COVID-19. ORF8 specifically targets CD14 + monocytes to produce pro-inflammatory cytokines through NLRP3 mediated inflammasomal activation. Importantly, ORF8 protein level in the blood of patients correlates with severity and disease-specific mortality in with acute SARS-CoV-2 infection. The ORF8-induced inflammasome response was readily inhibited by the NLRP3 inhibitor MCC950 in vitro. Our study identifies a dominant cause of pathogenesis, its underlying mechanism, and a potential new treatment for severe COVID-19 infection. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

H7N9 avian influenza virus infection in men is associated with testosterone depletion

Human infections with H7N9 avian influenza A virus that emerged in East China in 2013 and caused high morbidity rates were more frequently detected in men than in women over the last five epidemic waves. However, molecular markers associated with poor disease outcomes in men are still unknown. In this study, we systematically analysed sex hormone and cytokine levels in males and females with laboratory-confirmed H7N9 influenza in comparison to H7N9-negative control groups as well as laboratory-confirmed seasonal H1N1/H3N2 influenza cases (n = 369). Multivariable analyses reveal that H7N9-infected men present with considerably reduced testosterone levels associated with a poor outcome compared to non-infected controls. Regression analyses reveal that testosterone levels in H7N9-infected men are negatively associated with the levels of several pro-inflammatory cytokines, such as IL-6 and IL-15. To assess whether there is a causal relationship between low testosterone levels and avian H7N9 influenza infection, we used a mouse model. In male mice, we show that respiratory H7N9 infection leads to a high viral load and inflammatory cytokine response in the testes as well as a reduction in pre-infection plasma testosterone levels. Collectively, these findings suggest that monitoring sex hormone levels may support individualized management for patients with avian influenza infections.

Burn-injured skin is marked by a prolonged local acute inflammatory response of innate immune cells and pro-inflammatory cytokines.

The systemic and local immune response in burn patients is often extreme and derailed. As excessive inflammation can damage healthy tissues and slow down the healing process, modulation of inflammatory responses could limit complications and improve recovery. Due to its complexity, more detailed information on the immune effects of thermal injury is needed to improve patient outcomes. We therefore characterized and quantified subsets of immune cells and mediators present in human burn wound tissue (eschar), sampled at various time points. This study shows that after burn injury, the number of immune cells were persistently increased, unlike the normal wound healing process. There was an immediate, strong increase in neutrophils and a moderate increase in monocytes/macrophages and lymphocytes, especially in the second and third week post burn. The percentage of classical (CD14highCD16-) monocytes/macrophages demonstrated a steady decrease over time, whereas the proportion of intermediate (CD14highCD16+) monocytes/macrophages slowly increased. The absolute numbers of T cells, NK cells and B cells increased up to week 3, while the fraction of γδ T cells was increased only in week 1. Secretome profiling revealed high levels of chemokines and an overall pro-inflammatory cytokine milieu in burn tissue. The local burn immune response shows similarities to the systemic immune reaction, but differs in neutrophil maturity and lymphocyte composition. Altogether, the neutrophil surges, high levels of pro-inflammatory cytokines and limited immunosuppression might be key factors that prolong the inflammation phase and delay the wound healing process in burns.