Leukocyte Response Research Articles

Mitochondrial respiratory capacity modulates LPS-induced inflammatory signatures in human blood

Mitochondria modulate inflammatory processes in various model organisms, but it is unclear how much mitochondria regulate immune responses in human blood leukocytes. Here, we examine the effect of i) experimental perturbations of mitochondrial respiratory chain function, and ii) baseline inter-individual variation in leukocyte mitochondrial energy production capacity on stimulated cytokine release and glucocorticoid (GC) sensitivity. In a first cohort, whole blood from 20 healthy women and men was stimulated with increasing concentrations of the immune agonist lipopolysaccharide (LPS). Four inhibitors of mitochondrial respiratory chain Complexes I, III, IV, and V were used (LPS ​+ ​Mito-Inhibitors) to acutely perturb mitochondrial function, GC sensitivity was quantified using the GC-mimetic dexamethasone (DEX) (LPS ​+ ​DEX), and the resultant cytokine signatures mapped with a 20-cytokine array. Inhibiting mitochondrial respiration caused large inter-individual differences in LPS-stimulated IL-6 reactivity (Cohen’s d ​= ​0.72) and TNF-α (d ​= ​1.55) but only minor alteration in EC50-based LPS sensitivity (d ​= ​0.21). Specifically, inhibiting mitochondrial Complex IV potentiated LPS-induced IL-6 levels by 13%, but inhibited TNF-α induction by 72%, indicating mitochondrial regulation of the IL-6/TNF-α ratio. As expected, DEX treatment suppressed multiple LPS-induced pro-inflammatory cytokines (IFN-γ, IL-6, IL-8, IL-1β, TNF-α) by >85% and increased the anti-inflammatory cytokine IL-10 by 80%. Inhibiting Complex I potentiated DEX suppression of IL-6 by a further 12% (d ​= ​0.73), indicating partial mitochondrial modulation of glucocorticoid sensitivity. Finally, to examine if intrinsic mitochondrial respiratory capacity may explain a portion of immune reactivity differences across individuals, we measured biochemical respiratory chain enzyme activities and mitochondrial DNA copy number in isolated peripheral blood mononuclear cells (PBMCs) from a second cohort of 44 healthy individuals in parallel with LPS-stimulated IL-6 and TNF-α response. Respiratory chain function, particularly Complex IV activity, was positively correlated with LPS-stimulated IL-6 levels (r ​= ​0.45, p ​= ​0.002). Overall, these data provide preliminary evidence that mitochondrial behavior modulates LPS-induced inflammatory cytokine signatures in human blood.

Mda5/MAVS are essential for host resistance against Aspergillus fumigatus

Abstract Type I and III interferons act as important activators of antifungal neutrophil response in the lungs. RIG-I like receptors (RLR) are cytosolic RNA sensors that signal through the MAVS adaptor in order to activate interferon responses against viruses. Whether this pattern-recognition receptor family has broader effects on host immunity against other pathogen families remains to be fully explored. Herein we demonstrate that Mda5/MAVS signaling was essential for host resistance against pulmonary Aspergillus fumigatus challenge through the regulation of antifungal leukocyte responses. Interesting, induction of type I interferons after A. fumigatus challenge was only partially dependent on Mda5/MAVS signaling, while type III interferon expression was entirely dependent on Mda5/MAVS signaling. Ultimately, type I and III interferon signaling drove the expression of CXCL10, which is critical in resistance against invasive aspergillosis in transplant patients. Importantly, we found that polymorphisms in Ifih1 and Mavs were associated with the incidence of invasive pulmonary aspergillosis in a human HSCT cohort. Moreover, polymorphisms in the Ifih1 gene alter the inflammatory response induced in patients with invasive pulmonary aspergillosis, which include interferon-responsive chemokines. In conclusion, our data broaden the role of the RLR family in the regulating innate immunity during infection to include a role in immunity against Aspergillus fumigatus in both mice and humans.

Complex interactions between bacteria and haemosporidia in coinfected hosts: An experiment.

Hosts are typically coinfected by multiple parasite species whose interactions might be synergetic or antagonistic, producing unpredictable physiological and pathological impacts on the host. This study shows the interaction between Plasmodium spp. and Leucocytozoon spp. in birds experimentally infected or not infected with Mycoplasma gallisepticum.In 1994, the bacterium Mycoplasma gallisepticum jumped from poultry to wild birds in which it caused a major epidemic in North America. Birds infected with M. gallisepticum show conjunctivitis as well as increased levels of corticosterone.Malaria and other haemosporidia are widespread in birds, and chronic infections become apparent with the detectable presence of the parasite in peripheral blood in response to elevated levels of natural or experimental corticosterone levels.Knowing the immunosuppressive effect of corticosterone on the avian immune system, we tested the hypothesis that chronic infections of Plasmodium spp. and Leucocytozoon spp. in house finches would respond to experimental inoculation with M. gallisepticum as corticosterone levels are known to increase following inoculation. Plasmodium spp. infection intensity increased within days of M. gallisepticum inoculation as shown both by the appearance of infected erythrocytes and by the increase in the number and the intensity of positive PCR tests. Leucocytozoon spp. infection intensity increased when Plasmodium spp. infection intensity increased, but not in response to M. gallisepticum inoculation. Leucocytozoon spp. and Plasmodium spp. seemed to compete in the host as shown by a negative correlation between the changes in their PCR score when both pathogens were present in the same individual.Host responses to coinfection with multiple pathogens measured by the hematocrit and white blood cell count depended on the haemosporidian community composition. Host investment in the leukocyte response was higher in the single‐haemosporidia‐infected groups when birds were infected with M. gallisepticum.A trade‐off was observed between the immune control of the chronic infection (Plasmodium spp./Leucocytozoon spp.) and the immune response to the novel bacterial infection (M. gallisepticum).

Mesenchymal Stromal Cell-Derived Small Extracellular Vesicles Induce Ischemic Neuroprotection by Modulating Leukocytes and Specifically Neutrophils.

Background and Purpose- Small extracellular vesicles (sEVs) obtained from mesenchymal stromal cells (MSCs) were shown to induce neurological recovery after focal cerebral ischemia in rodents and to reverse postischemic lymphopenia in peripheral blood. Since peripheral blood cells, especially polymorphonuclear neutrophils (PMNs), contribute to ischemic brain injury, we analyzed brain leukocyte responses to sEVs and investigated the role of PMNs in sEV-induced neuroprotection. Methods- Male C57Bl6/j mice were exposed to transient intraluminal middle cerebral artery occlusion. After reperfusion, vehicle or sEVs prepared from conditioned media of MSCs raised from bone marrow samples of 3 randomly selected healthy human donors were intravenously administered. sEVs obtained from normoxic and hypoxic MSCs were applied. PMNs were depleted in vehicle and MSC-sEV-treated mice. Neurological deficits, ischemic injury, blood-brain barrier integrity, peripheral blood leukocyte responses, and brain leukocyte infiltration were evaluated over 72 hours. Results- sEV preparations of all 3 donors collected from normoxic MSCs significantly reduced neurological deficits. Preparations of 2 of these donors significantly decreased infarct volume and neuronal injury. sEV-induced neuroprotection was consistently associated with a decreased brain infiltration of leukocytes, namely of PMNs, monocytes/macrophages, and lymphocytes. sEVs obtained from hypoxic MSCs (1% O2) had similar effects on neurological deficits and ischemic injury as MSC-sEVs obtained under regular conditions (21% O2) but also reduced serum IgG extravasation-a marker of blood-brain barrier permeability. PMN depletion mimicked the effects of MSC-sEVs on neurological recovery, ischemic injury, and brain PMN, monocyte, and lymphocyte counts. Combined MSC-sEV administration and PMN depletion did not have any effects superior to PMN depletion in any of the readouts examined. Conclusions- Leukocytes and specifically PMNs contribute to MSC-sEV-induced ischemic neuroprotection. Individual MSC-sEV preparations may differ in their neuroprotective activities. Potency assays are urgently needed to identify their therapeutic efficacy before clinical application. Visual Overview- An online visual overview is available for this article.

Transient ischemic stroke induces substantial leukocyte recruitment to both the ischemic and non‐ischemic cerebrovasculature as visualized through live animal imaging.

Ischemic stroke is a leading cause of a death and disability worldwide. Current treatment strategies rely on early restoration of blood flow; however, injury persists despite successful reperfusion. The innate immune response plays a pivotal role in reperfusion injury, but the mechanisms and time‐course of neutrophil and monocyte recruitment to the cerebrovasculature (CBV) during ischemic stroke remain unclear. We sought to characterize the real‐time leukocyte response at the CBV in a mouse stroke model with reperfusion.To visualize leukocyte recruitment induced by ischemia, Ly6G‐tdTomato mice (which have fluorescent red neutrophils) were subjected to 90 minutes of transient middle cerebral artery occlusion (tMCAO) followed by reperfusion. Real‐time spinning‐disc confocal intravital microscopy of the CBV was performed at 24 and 72 hours following reperfusion. Intracranial windows were placed in both ischemic and non‐ischemic hemispheres; and tMCAO treated mice were compared to mice that underwent sham surgery. Neutrophil recruitment (adhesion, rolling) and extravasation were quantified in vivo on both the ischemic and contralateral non‐ischemic hemispheres. Postmortem staining with 2,3,5‐triphenyltetrazolium chloride was used to confirm induction of the stroke and to quantify infarct volume. In addition, wide‐field microscopy was used to examine neutrophil extravasation across the parenchyma.Animals subjected to sham surgery showed negligible leukocyte recruitment to the CBV. However, marked increases in leukocyte rolling, adhesion, and extravasation were observed at 24 and 72 hours following the tMCAO procedure. Interestingly, non‐ischemic hemispheres also demonstrated increased leukocyte rolling and adhesion, but not extravasation tMCAO group. Also, substantially more neutrophil extravasation was observed in the core of the infarct at 72 hours. These findings are consistent with an early recruitment of neutrophils to the cortical surface at early time points and a delayed infiltration into the core, as reported by others.These results show that a significant leukocyte response persists after reperfusion following tMCAO. This response is present both on the ischemic and, to a lesser extent, the non‐ischemic cortical CBV. Acute leukocyte recruitment observed on the non‐ischemic hemisphere suggests that ischemic stroke triggers a global inflammatory response within the CBV. Further reducing inflammation through immunomodulatory therapy in conjunction with reperfusion therapy may be beneficial in not only reducing stroke volume but potentially mitigating inflammation mediated secondary neuronal damage.Support or Funding InformationNIH R01 HL046849 and R37 HL064774 to W.A.M.

Changes in urinary bladder structure and systemic inflammation response following incomplete transection versus contusion spinal cord injury in rat model

Objective- The current study was conducted to evaluate changes in the urinary bladder structure and leukocyte profile as an important index of the systemic inflammation response for two different types of spinal cord injury (SCI) in a rat model. Design- Experimental Study.Animals- Forty adult healthy female Sprague-Dawley rats.Procedure- Animals were assigned into two equal model groups: the incomplete transection group (ITG) and the contusion group (CG). In both groups, SCI was created at the T9-10 level of the column. Alterations in the urinary bladder construction and changes in the leukocytes were examined in both groups post-surgically. Results- Degenerative changes and a reduction in the cellular volume in the mucous layer, hyperemia, and the presence of inflammatory cells in the submucosa were the most important findings in both SCI groups. The extent of the destructive lesions was more prominent in the CG 14 days after operation. At 28 days after surgery, pathological lesions including leukocyte infiltration in the submucosa, denudation of the urothelial mucosa, severe edema, atrophy of the muscle layer, and necrosis of muscle fibers in some areas were recorded in both groups; the extent and severity of the lesions were more evident in the CG. There was no significant difference between the white blood cells and N/L ratio at the different times in the CG and ITG groups. Conclusion and Clinical Relevance- Despite the similar leukocyte response in the IGT and CG, more severe degenerative histological alternations in the urinary bladder structure were observed in the CG. Therefore, attention should be paid to the extent of cystitis in these patients in clinical trials and interventions.

Differential effects of BCG vaccine on immune responses induced by vi polysaccharide typhoid fever vaccination: an explorative randomized trial

The Vi polysaccharide typhoid fever vaccine (TFV) provides incomplete protection against typhoid fever. BCG, the vaccine against tuberculosis, can potentiate immune responses to other vaccines through induction of trained innate immunity and heterologous adaptive immunity. We performed an explorative, randomized, noncontrolled open trial to investigate whether BCG vaccination increases humoral and cellular response to TFV and whether BCG and TFV modulate nonspecific immune responses. Thirty volunteers were randomized to receive either TFV alone or BCG followed by TFV after 2 weeks. Ex vivo leukocyte responses and anti-Vi IgG antibody titers were measured 2 weeks and 3 months after TFV. BCG administration prior to TFV vaccination did not increase specific humoral or cellular immune responses to Salmonella typhi. TFV vaccination decreased pro-inflammatory responses to non-related stimuli. This effect was counteracted by prior BCG administration, which also led to decreased IL-10 and increased IL-22 responses to non-related stimuli. In an in vitro model of trained immunity TFV led to immunotolerance, which was partially reversed by BCG-induced trained immunity. BCG does not modulate adaptive immune responses to TFV but partially prevents inhibition of innate immune responses induced by TFV. Nonspecific effects of vaccines to unrelated microbial stimuli must be considered in the evaluation of their biological effects (ClinicalTrials.gov NCT02175420).

Patient-specific factors modulate leukocyte response in dimethyl fumarate treated MS patients

ObjectiveDetermine if patient-specific factors modulate absolute lymphocyte count (ALC), neutrophil count (ANC), and/or Neutrophile-lymphocyte ratio (NLR) in Dimethyl Fumarate (DMF) treated patients.MethodsA retrospective study of patients who initiated DMF between 2013–2018. A multicenter study of two MS clinics: Charlottesville, VA (UVA) and Dallas, TX (DaVA)Results103 patients (67-UVA, 36-DaVA) met eligibility. At baseline, the DaVa population was younger (mean±sd: 38.6±9.0 vs 42.2±12.5, p 0.152) and had a higher proportion of males (61% vs. 35%), consistent with a veteran cohort. Pre-treatment, all other laboratory parameters were similar between the two groups. On treatment there was a 30% lowering of mean ALC, with 3% having grade-3 lymphopenia (ALC < 500). Sustained neutropenia occurred in 3.9% of patients and was more common in males. Over 50% of patients had a high NLR at baseline, with a further 44% increase in NLR on-treatment. Age was significantly predictive of lymphopenia, with grade-3 lymphopenia found in 33% of patients ≥ 55 years. Neutropenia was more common in males. Serum BG (sBG) has modest correlation to leukocyte parameters. BMI was not correlated with any leukocyte-related outcomes.ConclusionsPatient-specific factors, specifically–age, sex, and serum blood glucose, modulate leukocyte response and ratios in DMF treated MS patients. Age appears to be a relevant predictor of lymphopenia and should be a factor in treatment decision making. Neutropenia, independent of lymphopenia, can occur and males may be at increased risk. High sBG may impact leukocyte count and ratios in MS patients and merits further study, particularly in patients with diabetes. NLR is abnormal in MS and increased with DMF-treatment, the clinical implications of this will require further study.

Intravital Microscopy of the Beating Murine Heart to Understand Cardiac Leukocyte Dynamics.

Cardiovascular disease is the leading cause of worldwide mortality. Intravital microscopy has provided unprecedented insight into leukocyte biology by enabling the visualization of dynamic responses within living organ systems at the cell-scale. The heart presents a uniquely dynamic microenvironment driven by periodic, synchronous electrical conduction leading to rhythmic contractions of cardiomyocytes, and phasic coronary blood flow. In addition to functions shared throughout the body, immune cells have specific functions in the heart including tissue-resident macrophage-facilitated electrical conduction and rapid monocyte infiltration upon injury. Leukocyte responses to cardiac pathologies, including myocardial infarction and heart failure, have been well-studied using standard techniques, however, certain questions related to spatiotemporal relationships remain unanswered. Intravital imaging techniques could greatly benefit our understanding of the complexities of in vivo leukocyte behavior within cardiac tissue, but these techniques have been challenging to apply. Different approaches have been developed including high frame rate imaging of the beating heart, explantation models, micro-endoscopy, and mechanical stabilization coupled with various acquisition schemes to overcome challenges specific to the heart. The field of cardiac science has only begun to benefit from intravital microscopy techniques. The current focused review presents an overview of leukocyte responses in the heart, recent developments in intravital microscopy for the murine heart, and a discussion of future developments and applications for cardiovascular immunology.

Spleen tyrosine kinase inhibition is an effective treatment for established vasculitis in a pre-clinical model

The anti-neutrophil cytoplasm antibody (ANCA)-associated vasculitides (AAV) are a group of life-threatening multi-system diseases characterized by necrotising inflammation of small blood vessels and crescentic glomerulonephritis. ANCA are thought to play a direct pathogenic role. Previous studies have shown that spleen tyrosine kinase (SYK) is phosphorylated during ANCA-induced neutrophil activation in vitro. However, the role of SYK in vivo is unknown. Here, we studied its role in the pathogenesis of experimental autoimmune vasculitis, a pre-clinical model of myeloperoxidase-ANCA-induced pauci-immune systemic vasculitis in the Wistar Kyoto rat. Up-regulation of SYK expression in inflamed renal and pulmonary tissue during early autoimmune vasculitis was confirmed by immunohistochemical and transcript analysis. R406, the active metabolite of fostamatinib, a small molecule kinase inhibitor with high selectivity for SYK, inhibited ANCA-induced pro-inflammatory responses in rat leucocytes in vitro. In an in vivo study, treatment with fostamatinib for 14 days after disease onset resulted in rapid resolution of urinary abnormalities, significantly improved renal and pulmonary pathology, and preserved renal function. Short-term exposure to fostamatinib did not significantly affect circulating myeloperoxidase-ANCA levels, suggesting inhibition of ANCA-induced inflammatory mechanisms in vivo. Finally, SYK expression was demonstrated within inflammatory glomerular lesions in ANCA-associated glomerulonephritis in patients, particularly within CD68+ve monocytes/macrophages. Thus, our data indicate that SYK inhibition warrants clinical investigation in the treatment of AAV.

Changes in redox and endoplasmic reticulum homeostasis are related to congenital generalized lipodystrophy type 2

CGL type 2 is a rare autosomal recessive syndrome characterized by an almost complete lack of body fat. CGL is caused by loss-of-function mutations in both alleles of the BSCL2 gene that codifies to seipin. Subjects often show hyperglycemia, decreased HDL-c, and hypoadiponectinemia. These laboratory findings are important triggers for changes in redox and ER homeostasis. Therefore, our aim was to investigate whether these intracellular mechanisms are associated with this syndrome. We collected blood from people from Northeastern Brazil with 0, 1, and 2 mutant alleles for the rs786205071 in the BSCL2 gene. Through the qPCR technique, we evaluated the expression of genes responsible for triggering the antioxidant response, DNA repair, and ER stress in leukocytes. Colorimetric tests were applied to quantify lipid peroxidation and to evaluate the redox status of glutathione, as well as to access the panorama of energy metabolism. Long extension PCR was performed to observe leukocyte mitochondrial DNA lesions, and the immunoblot technique to investigate plasma adiponectin concentrations. Subjects with the rs786205071 in both BSCL2 alleles showed increased transcription of NFE2L2, APEX1, and OGG1 in leukocytes, as well as high concentrations of malondialdehyde and the GSSG:GSH ratio in plasma. We also observed increase of mitochondrial DNA lesions and XBP1 splicing, as well as a decrease in adiponectin and HDL-c. Our data suggest the presence of lipid lesions due to changes in redox homeostasis in that group, associated with increased levels of mitochondrial DNA damage and transcriptional activation of genes involved with antioxidant response and DNA repair.

DNA damage response in peripheral mouse blood leukocytes in vivo after variable, low-dose rate exposure.

Environmental contamination and ingestion of the radionuclide Cesium-137 (137Cs) is a large concern in fallout from a nuclear reactor accident or improvised nuclear device, and highlights the need to develop biological assays for low-dose rate, internal emitter radiation. To mimic low-dose rates attributable to fallout, we have developed a VAriable Dose-rate External 137Cs irradiatoR (VADER), which can provide arbitrarily varying and progressive low-dose rate irradiations in the range of 0.1-1.2Gy/day, while circumventing the complexities of dealing with radioactively contaminated biomaterials. We investigated the kinetics of mouse peripheral leukocytes DNA damage response in vivo after variable, low-dose rate 137Cs exposure. C57BL/6 mice were placed in the VADER over 7days with total accumulated dose up to 2.7Gy. Peripheral blood response including the leukocyte depletion, apoptosis as well as its signal protein p53 and DNA repair biomarker γ-H2AX was measured. The results illustrated that blood leukocyte numbers had significantly dropped by day 7. P53 levels peaked at day 2 (total dose = 0.91Gy) and then declined; whereas, γ-H2AX fluorescence intensity (MFI) and foci number generally increased with accumulated dose and peaked at day 5 (total dose = 2.08Gy). ROC curve analysis for γ-H2AX provided a good discrimination of accumulated dose < 2Gy and ≥ 2Gy, highlighting the potential of γ-H2AX MFI as a biomarker fordosimetry in a protracted, environmental exposure scenario.

The actin-bundling protein L-plastin-A double-edged sword: Beneficial for the immune response, maleficent in cancer.

The dynamic organization of the actin cytoskeleton into bundles and networks is orchestrated by a large variety of actin-binding proteins. Among them, the actin-bundling protein L-plastin is normally expressed in hematopoietic cells, where it is involved in the immune response. However, L-plastin is also often ectopically expressed in malignant cancer cells of non-hematopoietic origin and is even considered as a marker for cancer progression. Post-translational modification modulates L-plastin activity. In particular, L-plastin Ser5 phosphorylation has been shown to be important for the immune response in leukocytes as well as for invasion and metastasis formation of carcinoma cells. This chapter discusses the physiological and pathological role of L-plastin with a special focus on the importance of L-plastin Ser5 phosphorylation for the protein functions. The potential use of Ser5 phosphorylated L-plastin as a biomarker and/or therapeutic target will be evoked.

Intraoperative Cell Salvage as an Alternative to Allogeneic (Donated) Blood Transfusion: A Prospective Observational Evaluation of the Immune Response Profile

Allogeneic blood transfusion (ABT) is associated with transfusion-related immune modulation (TRIM) and subsequent poorer patient outcomes including perioperative infection, multiple organ failure, and mortality. The precise mechanism(s) underlying TRIM remain largely unknown. During intraoperative cell salvage (ICS) a patient’s own (autologous) blood is collected, anticoagulated, processed, and reinfused. One impediment to understanding the influence of the immune system on transfusion-related adverse outcomes has been the inability to characterize immune profile changes induced by blood transfusion, including ICS. Dendritic cells and monocytes play a central role in regulation of immune responses, and dysfunction may contribute to adverse outcomes. During a prospective observational study (n = 19), an in vitro model was used to assess dendritic cell and monocyte immune responses and the overall immune response following ABT or ICS exposure. Exposure to both ABT and ICS suppressed dendritic cell and monocyte function. This suppression was, however, significantly less marked following ICS. ICS presented an improved immune competence. This assessment of immune competence through the study of intracellular cytokine production, co-stimulatory and adhesion molecules expressed on dendritic cells and monocytes, and modulation of the overall leukocyte response may predict a reduction of adverse outcomes ( i.e., infection) following ICS.

The Mechanisms of Sijunzi Decoction in the Treatment of Chronic Gastritis Revealed by Network Pharmacology.

Chronic gastritis is characterized by inflammation in the gastric mucosa with a vicious circle in inflammatory cells and inflammatory mediators. Stomach adenocarcinoma would occur in the metaplastic gastric mucosa of chronic gastritis. Sijunzi decoction is a famous classical formula for the treatment of chronic gastritis. Although previous studies revealed some functions of Sijunzi decoction in treating chronic gastritis, the underlying mechanisms have not been illustrated clearly. In this study, we used network pharmacology to investigate the mechanism of Sijunzi decoction in treating chronic gastritis. Firstly, online datasets TCMSP, SWISS, and DisGeNET were used to investigate the functional mechanism of Sijunzi decoction against chronic gastritis and 18 genes were identified as targets of Sijunzi decoction in chronic gastritis. These 18 genes can be categorized into immunologically related genes and cancer-related genes. GO analysis showed that the 18 target genes were mainly enriched in angiogenesis, nitric oxide biosynthetic process, ERK1 and ERK2 cascade, cellular response to drug, and MAPK cascade. So, Sijunzi decoction alleviated chronic gastritis by inhibiting the local inflammatory response. Furthermore, we also investigated the impact of Sijunzi decoction on the peripheral blood leukocytes with our own RNA sequencing (RNA-seq) data of 11 chronic superficial gastritis patients. 102 differentially expressed genes (DEGs) were identified by comparing RNA-seq data of chronic superficial gastritis patients with healthy control groups. After performing a functional analysis on 102 DEGs and Sijunzi decoction potential targets and taking the intersection of these pathways, we found that platelet activation, angiogenesis, and pathways in cancer were candidate target pathways regulated by Sijunzi decoction. Thus, Sijunzi decoction also alleviates chronic gastritis by suppressing inflammatory response of peripheral blood leukocytes. Our results showed that Sijunzi decoction can ameliorate the local gastric inflammation and inflammations in peripheral blood leukocytes and might also reduce the incidence of stomach cancer in chronic gastritis.

Platelets Modulate IFN-γ Production against Candida albicans in Peripheral Blood Mononuclear Cells via Prostaglandins.

Platelets are known to have immunomodulatory properties. They modulate immune responses of leukocytes against various pathogens, including fungi. Candida albicans can cause systemic infection in immunocompromised individuals that is associated with a high mortality and morbidity. In the current study, we explored the role of platelets in antifungal host defense against C. albicans PBMCs were stimulated with heat-killed (HK) C. albicans in the presence or absence of isolated washed platelets. Cytokines were quantified from culture supernatants by ELISA. Inhibition of platelet receptors and cytokine pathways were used to elucidate the mechanisms involved in platelet-leukocyte interaction. In the presence of platelets, PBMCs produced less IFN-γ upon stimulation with HK C. albicans This effect was dependent on the direct contact between platelets and leukocytes but was independent of the platelet GPIb and P-selectin receptors. The attenuation of IFN-γ was not a direct effect on T cells but was dependent on the presence of APC and T cells. Platelets did not modulate the Th-1-polarizing cytokines IL-12 and IL-18. The addition of PG (PGE2) further diminished IFN-γ levels in PBMCs, and supplementation of cells with nonsteroidal anti-inflammatory drugs was able to restore the level of IFN-γ. Overall, we show that modulation of the Th1 response against C. albicans by platelets is dependent on PGs.

Effect of pre-supplementation with Pleurotus sajor-caju crude extracts on body weight and consequence responses of leukocytes and immune organs in fancy carp following inoculation with Aeromonas veronii.

Aim:The present study aimed at highlighting the effects of oyster mushroom (Pleurotus sajor-caju), as a dietary supplement on growth performance, differential leukocytes population, and histological changes of melanomacrophage centers (MMCs) in spleen and kidney of fancy carp on bacterial infection.Materials and Methods:A total of 60 fancy carp were allocated into four groups according to feed formulations including; (1) basal diet with 2% crude extract of P. sajor-caju, (2) basal diet with 2% b-glucan, whereas Group 3, and Group 4 were positive and negative control, which were fed only basal diet. Diets were provided for 30 days, thereafter, fish of Group 1 to Group 3 were intraperitoneally injected with Aeromonas veronii (1.8×109 CFU), whereas Group 4 was injected with normal saline. At day 7 post-bacterial inoculation, all fish were weighed, whole blood was collected for differential white blood cell count, and two visceral organs, posterior kidney and spleen, were collected from euthanized fish to observe histological changes, particularly MMCs.Results:No significant differences in body weight were found (p>0.05) at 1st week of the experiment; however, fish body weight was significantly increased from week 2 to week 4 of the experiment. Increased monocyte number was found in carp fish fed with the P. sajor-caju or b-glucan supplemented diets compared to the control groups (p<0.05). The proliferation of monocyte in fish was consistent with increased number and size of MMCs in hemotopoietic organs, posterior kidney and spleen, especially in fancy carp fed with of P. sajor-caju crude extract and commercially available b-glucan before bacterial inoculation in fish.Conclusion:These findings indicate that crude polysaccharide from P. sajor-caju can be potentially used as a feed additive that might promote innate immune function in fish.

MEK1 regulates pulmonary macrophage inflammatory responses and resolution of acute lung injury.

The MEK1/2-ERK1/2 pathway has been implicated in regulating the inflammatory response to lung injury and infection, and pharmacologic MEK1/2 inhibitor compounds are reported to reduce detrimental inflammation in multiple animal models of disease, in part through modulation of leukocyte responses. However, the specific contribution of myeloid MEK1 in regulating acute lung injury (ALI) and its resolution remain unknown. Here, the role of myeloid Mek1 was investigated in a murine model of LPS-induced ALI (LPS-ALI) by genetic deletion using the Cre-floxed system (LysMCre × Mekfl), and human alveolar macrophages from healthy volunteers and patients with acute respiratory distress syndrome (ARDS) were obtained to assess activation of the MEK1/2-ERK1/2 pathway. Myeloid Mek1 deletion results in a failure to resolve LPS-ALI, and alveolar macrophages lacking MEK1 had increased activation of MEK2 and the downstream target ERK1/2 on day 4 of LPS-ALI. The clinical significance of these findings is supported by increased activation of the MEK1/2-ERK1/2 pathway in alveolar macrophages from patients with ARDS compared with alveolar macrophages from healthy volunteers. This study reveals a critical role for myeloid MEK1 in promoting resolution of LPS-ALI and controlling the duration of macrophage proinflammatory responses.

Substance P blockade effects on peripheral blood leukocytic reaction in secondary chronic inflammation

In an experimental prospective controlled study the leukocyte response of peripheral blood on the background of blockade of receptors of substance P was studied on the carrageenan model of secondary chronic inflammation in 132 rats. Compared with the natural course of inflammation, at 6 hours there was a significant decrease in total white blood cells count by 1.28 (p&lt;0.05), as well as a significant decrease in the number of polymorphonuclear neutrophils by 4 times (p&lt;0.05) and the number of eosinophils by 3 times (p&lt;0.05) by 10 days. On day 28, there was a significant increase in the number of monocytes in 2 times (p&lt;0,01). The results of the study indicate that in natural conditions of inflammation, substance P stimulates blood leukocytes, as well as prolongs the inflammatory response, and its blocking helps to reduce the inflammatory response.

Spebrutinib (CC-292) Affects Markers of B Cell Activation, Chemotaxis, and Osteoclasts in Patients with Rheumatoid Arthritis: Results from a Mechanistic Study.

IntroductionSpebrutinib (CC-292) is an orally administered, covalent, small-molecule inhibitor of Bruton’s tyrosine kinase (BTK), part of the B-cell and Fc receptor signaling pathways. This study evaluated spebrutinib pharmacology and mechanism of action over a 4-week treatment period in patients with active rheumatoid arthritis (RA).MethodsPrimary human B cells, T cells, natural killer cells, macrophages, dendritic cells, basophils, and osteoclasts were treated with spebrutinib in vitro. Clinical pharmacodynamics were studied in 47 patients with active RA on background methotrexate therapy randomized to oral spebrutinib 375 mg/day or placebo.ResultsIn vitro, spebrutinib inhibited B-cell proliferation more potently than T-cell proliferation and reduced both lymphoid and myeloid cytokine production and degranulation, as well as osteoclastogenesis. Clinical efficacy trended higher in spebrutinib-treated RA patients, with 41.7% (10/24) achieving ≥ 20% improvement in ACR response criteria (ACR20) versus 21.7% (5/23) of placebo patients at week 4 (P = 0.25). Treatment-emergent adverse events were comparable between treatment groups. In spebrutinib-treated patients, median BTK occupancy in peripheral blood was 83%, and significant increases in total CD19+ and mature-naive CD27−CD38−IgD+ B cells and decreases in transitional CD27−CD38+ B cells were observed. Spebrutinib significantly reduced serum chemokines chemokine ligand 13 (CXCL13), macrophage inflammatory protein-1β (MIP-1β), and the bone resorption biomarker carboxy-terminal collagen cross-linking telopeptide (CTX-I) (P < 0.05). Clinical response to spebrutinib was associated with lower increases in CD19+ B cells and greater decreases in CXCL13 and MIP-1β from baseline to week 4. High CD19+ B cells and low CTX-I at baseline were associated with better spebrutinib clinical response.ConclusionsSpebrutinib inhibited various leukocyte responses in vitro, including those of B cells and osteoclasts. In this small study in RA patients, spebrutinib was well tolerated, showed a downward trend for symptoms, significantly modulated B-cell populations, and reduced markers of chemotaxis and osteoclast activity.Trial RegistrationNCT01975610.Electronic Supplementary MaterialThe online version of this article (10.1007/s40744-019-00182-7) contains supplementary material, which is available to authorized users.