MIP-2 Levels Research Articles

Role of pyroptosis-related cytokines in the prediction of lung cancer

ObjectivesLung cancer is the leading cause to induce cancer-related mortality. Effective biomarkers for prediction the occurrence of lung cancer is urgently needed. Our previous studies indicated that pyroptosis-related cytokines TNF-α, IFN-γ, MIP-1α, MIP-1β, MIP-2 and IP-10 is important to influence the efficacy of chemotherapy drug in lung cancer tissues. But the role of pyroptosis-related cytokines in prediction the occurrence of lung cancer is still unknown. MethodsBlood samples were collected from 258 lung cancer patients at different stage and 80 healthy volunteers. Serum levels of pyroptosis-related cytokines including TNF-α, IFN-γ, MIP-1α, MIP-1β, MIP-2 and IP-10 were measured by Cytometric Bead Array (CBA). ROC curve was performed to evaluate the cut-off value and diagnosis value for prediction and diagnosis of lung cancer. ResultsCompared with control group, the levels of IP-10, MIP-1α, MIP-1β, MIP-2 and TNF-α were significantly higher in lung cancer patients (45.5 (37.1–56.7): 57.2 (43.0–76.5), 34.4 (21.8–75.2): 115.4 (96.6–191.2), 49.3 (25.6–78.7): 160.5 (124.9–218.6), 22.6 (17.8–31.2): 77.9 (50.1–186.5), 3.80 (2.3–6.2): 10.3 (5.7–16.6)), but the level of IFN-γ was decreased in the patients (12.38 (9.1–27.8): 5.9 (3.5–9.7)). All the above cytokines were significantly associated with the diagnosis of lung cancer, and the AUC values of IFN-γ, IP-10, MIP-1α, MIP-1β, MIP-2, and TNF-α were 0.800, 0.656, 0.905, 0.921, 0.914, and 0.824. And the AUC can rise to 0.986 after combining the above factors, and the sensitivity and specificity also up to 96.7 % and 93.7 %, respectively. Additionally, TNF-α (r = 0.400, P < 0.01), MIP-2 (r = 0.343, P < 0.01), MIP-1α (r = 0.551, P < 0.01) and MIP-1β (r = 0.403, p < 0.01) were positively associated with occurrence of lung cancer, but IFN-γ (r = −0.483, p < 0.01) was negatively associated with occurrence of lung cancer. As far as the potential of early diagnosis of lung cancer, TNF-α (AUC = 0.577), MIP-1α (AUC = 0.804) and MIP-1β (AUC = 0.791) can predict the early stage of lung cancer, and combination of the above three cytokines has a better predictive efficiency (AUC = 0.854). ConclusionOur study establishes a link between the levels of IP-10, MIP-1α, MIP-1β, MIP-2, TNF-α and IFN-γ and diagnosis of lung cancer. Besides, we observed a synergistic effect of these five pyroptosis-related cytokines in diagnosing lung cancer patient, suggesting their potential as biomarkers for lung cancer diagnosis. Moreover, the combination of TNF-α, MIP-1α and MIP-1β are also potential predictors for the early diagnosis of lung cancer.

Airway epithelial cell-specific deletion of EGFR in Scnn1b transgenic mice modulates inflammatory responses but doesn’t affect muco-obstructive features of lung disease

Abstract Epidermal growth factor receptor (EGFR) is known to regulate airway mucous cell metaplasia, but the roles of EGFR-mediated pathway in the manifestation of cystic fibrosis (CF)-relevant lung phenotypes remain unclear. We hypothesized that airway epithelial cell-specific deficiency of EGFR mitigates muco-obstructive and inflammatory responses in sodium channel, non-voltage gated 1, beta subunit (Scnn1b) overexpressing -transgenic (Tg+) mice, a mouse model of human CF-like lung disease. To test our hypothesis, we generated airway epithelial cell-specific EGFR-deficient mice and performed various endpoint analyses. The deletion of EGFR in airway epithelial cells in Tg+ mice resulted in increased neutrophilic and macrophage infiltration into lung airspaces, which was accompanied by significantly increased levels of KC, G-CSF, MIP-2, MIP-1α, and MIP-1β. Additionally, as compared to EGFR-sufficient Tg+ mice, airway epithelial cell-specific EGFR-deficient Tg+ mice exhibited significantly increased postnatal mortality, compromised bacterial clearance and elevated markers of Th2 inflammation, including Il13, Slc26a4, and Retnla. Counterintuitively, the deletion of EGFR in airway epithelial cells of Tg+ mice did not alter mucous cell metaplasia, MUC5AC/5B production, and degree of mucus obstruction. Our data shows that, unlike inflammation, muco-obstruction-relevant responses in Tg+ mice are not dependent on airway epithelial cell-specific EGFR signaling.

Effects of myeloperoxidase on inflammatory responses with hypoxia in Citrobacter rodentium-infectious mice.

Myeloperoxidase (MPO)has been identified as a mediator in various inflammatory diseases. Bacterial infection of the intestine and hypoxia can both lead to inflammatory responses, but the role of MPO in these phenomena remains unclear. By building the MPO-/- mice, we evaluated relevant inflammatory factors and tissue damage in mice with intestinal Citrobacter rodentium infection and hypoxia. The body weight and excreted microorganisms were monitored. Intestinal tissues were collected 7 days after bacterial infection under hypoxia to undergo haematoxylin-eosin staining and assess the degree of pathological damage. ELISA assays were performed to quantify the serum levels of TNF-α, IFN-γ, IL-6, and IL-1β inflammatory cytokines. PCR, WB, and IF assays were conducted to determine the expression of chemokines MCP1, MIP2, and KC in the colon and spleen. The C. rodentium infection and hypoxia caused weight loss, intestinal colitis, and splenic inflammatory cells active proliferation in wild-type mice. MPO deficiency alleviated this phenomenon. MPO-/- mice also displayed a significant decline in bacteria clearing ability. The level of TNF-α in the serum and spleen was both lower in MPO-/- hypoxia C. rodentium-infected mice than that in wild-type mice. The chemokines expression levels of MIP2, KC,and MCP1 in the spleen and colon of each bacterial infected group were significantly increased (p < .05), while in hypoxia, the factors in the spleen and colon were decreased (p < .05). MPO deficiency was found to lower the levels of these chemokines compared with wild-type mice. MPO plays an important role of the inflammatory responses in infectious enteritis and hypoxia in mice, and the loss of MPO may greatly reduce the body's inflammatory responses to fight diseases.

A NOVEL OLIGONUCLEOTIDE MRNA MIMIC ATTENUATES HEMORRHAGE-INDUCED ACUTE LUNG INJURY.

Hemorrhagic shock (HS) is accompanied by a pronounced activation of the inflammatory response in which acute lung injury (ALI) is one of the most frequent consequences. Among the pivotal orchestrators of this inflammatory cascade, extracellular cold-inducible RNA-binding protein (eCIRP) emerges as a noteworthy focal point, rendering it as a promising target for the management of inflammation and tissue injury. Recently, we have reported that oligonucleotide poly(A) mRNA mimic termed A 12 selectively binds to the RNA binding region of eCIRP and inhibits eCIRP binding to its receptor TLR4. Furthermore, in vivo administration of eCIRP induces lung injury in healthy mice and that mouse deficient in CIRP showed protection from inflammation-associated lung injury. We hypothesize that A 12 inhibits systemic inflammation and ALI in HS. To test the impacts of A 12 on systemic and lung inflammation, extent of inflammatory cellular infiltration and resultant lung damage were evaluated in a mouse model of HS. Male mice were subjected to controlled hemorrhage with a mean arterial pressure of 30 mm Hg for 90 min and then resuscitated with Ringer's lactate solution containing phosphate-buffered saline (vehicle) or A 12 at a dose of 4 nmol/g body weight (treatment). The infusion volume was twice that of the shed blood. At 4 h after resuscitation, mice were euthanized, and blood and lung tissues were harvested. Blood and tissue markers of inflammation and injury were evaluated. Serum markers of injury (lactate dehydrogenase, alanine transaminase, and blood urea nitrogen) and inflammation (TNF-α, IL-6) were increased after HS and A 12 treatment significantly decreased their levels. A 12 treatment also decreased lung levels of TNF-α, MIP-2, and KC mRNA expressions. Lung histological injury score, neutrophil infiltration (Ly6G staining and myeloperoxidase activity), and lung apoptosis were significantly attenuated after A 12 treatment. Our study suggests that the capacity of A 12 in attenuating HS-induced ALI and may provide novel perspectives in developing efficacious pharmaceutics for improving hemorrhage prognosis.

Diterpene Coronarin Attenuates Lipopolysaccharide-Induced Acute Lung Injury in Both In Vivo and In Vitro Models.

Acute lung injury (ALI) is a clinical condition occurs due to severe systemic inflammatory response for clinical stimulus like pneumonia, sepsis, trauma, aspiration, inhalation of toxic gases, and pancreatitis. Disruption of alveolar barriers, activation of macrophages, infiltration of neutrophils, and proinflammatory cytokines are the vital events occurs during ALI. The drugs which inhibit these inflammatory response can protect lungs from inflammatory insults. In this study, we examined the potency of phytochemical coronarin, a diterpene which have been proven to possess anti-inflammatory, antioxidant, antiangiogenic, and antitumor activities. Healthy BALB/c mice were induced to acute lung injury with intra-tracheal administration of LPS and then treated with 5 and 10 mg/kg concentration of coronarin. The wet/dry lung weight of mice were estimated to assess the induction of pulmonary edema. BALF fluid was analyzed for protein concentrations and immune cells count. Myeloperoxidase activity and levels of chemokines MCP-2 and MIP-2, iNOS, COX-2, and PGE-2 were quantified to assess the immunomodulatory effect of coronarin against LPS-induced ALI. The levels of proinflammatory cytokines was measured to examine the anti-inflammatory property of coronarin, and it was confirmed with histopathological analysis of the lung tissue. Murine RAW 264.7 cells were utilized for the in vitro analysis. Cell cytoxicity and cytoprotective property of coronarin was assessed with MTT assay in LPS-treated Murine RAW 264.7. The anti-inflammatory property of coronarin was further confirmed in in vitro condition by estimating the levels of pro-inflammatory cytokines in coronarin-treated and untreated LPS-induced cells. Overall, our in vivo and in vitro results confirm coronarin significantly inhibited the infiltration of neutrophils prevented immunodulatory activity and synthesis of proinflammatory cytokines and alleviated the acute lung injury induced by LPS. Coronarin is a potent anti-inflammatory drug which can be subjected to further research to be prescribed as drug for ALI.

Lacking ARHGAP25 mitigates the symptoms of autoantibody-induced arthritis in mice.

Despite intensive research on rheumatoid arthritis, the pathomechanism of the disease is still not fully understood and the treatment has not been completely resolved. Previously we demonstrated that the GTPase-activating protein, ARHGAP25 has a crucial role in the regulation of basic phagocyte functions. Here we investigate the role of ARHGAP25 in the complex inflammatory process of autoantibody-induced arthritis. Wild-type and ARHGAP25 deficient (KO) mice on a C57BL/6 background, as well as bone marrow chimeric mice, were treated i.p. with the K/BxN arthritogenic or control serum, and the severity of inflammation and pain-related behavior was measured. Histology was prepared, leukocyte infiltration, cytokine production, myeloperoxidase activity, and superoxide production were determined, and comprehensive western blot analysis was conducted. In the absence of ARHGAP25, the severity of inflammation, joint destruction, and mechanical hyperalgesia significantly decreased, similarly to phagocyte infiltration, IL-1β, and MIP-2 levels in the tibiotarsal joint, whereas superoxide production or myeloperoxidase activity was unchanged. We observed a significantly mitigated phenotype in KO bone marrow chimeras as well. In addition, fibroblast-like synoviocytes showed comparable expression of ARHGAP25 to neutrophils. Significantly reduced ERK1/2, MAPK, and I-κB protein signals were detected in the arthritic KO mouse ankles. Our findings suggest that ARHGAP25 has a key role in the pathomechanism of autoantibody-induced arthritis in which it regulates inflammation via the I-κB/NF-κB/IL-1β axis with the involvement of both immune cells and fibroblast-like synoviocytes.

Docosahexaenoic acid protects against lipopolysaccharide-induced fetal growth restriction via inducing the ubiquitination and degradation of NF-κB p65 in placental trophoblasts

Lipopolysaccharide (LPS) could induce adverse birth outcomes by evoking inflammation. We investigated the effect and mechanism of docosahexaenoic acid (DHA) on LPS-induced placental inflammation and fetal growth restriction (FGR). In vivo, pregnant CD-1 mice were divided into four groups: Ctrl, DHA, LPS and DHA+LPS group. We found that DHA pretreatment reduced the incidence of FGR induced by LPS and activated the expression of peroxisome proliferators-activated receptor gamma (PPARγ) in placental tissue. Moreover, the LPS-induced increase of mRNA levels of Tnf-α, Il-6, Il-1β, Mip-2 and Kc in placental tissue was significantly attenuated by DHA pretreatment. A similar effect of DHA was observed in serum of pregnant mice and amniotic fluid. In contrast, the levels of the IL-10 were significantly increased after DHA pretreatment. In vitro, we clarified that DHA antagonized the activation of the NF-κB signaling pathway induced by LPS, which was dependent on PPARγ. Subsequently, CHX (translation inhibitor) was used to indicated that PPARγ significantly increased the degradation rate of p65, an effect that was inhibited by MG132 (proteasome inhibitor) treatment. Finally, it was confirmed that the activation of PPARγ could significantly promote the ubiquitination and degradation of p65. Our results suggested that DHA alleviated LPS-induced inflammatory responses and FGR by activating PPARγ expression, leading to p65 ubiquitination and degradation.

Effect of Huangdisan grain on improving cognitive impairment in VD rats and its mechanism in immune inflammatory response.

Vascular dementia (VD) is the second most common type of dementia after Alzheimer's disease (AD). Although the incidence rate is very high, there is no definitive treatment for VD. And it has serious impact on the quality of life of VD patients. In recent years, more and more studies about the clinical efficacy and pharmacological effects of traditional Chinese medicine (TCM) in the treatment of VD have been conducted. And Huangdisan grain has been used to treat VD patients with a good curative effect in clinic. This study was designed to investigate the effect of Huangdisan grain on the inflammatory response and cognitive function of VD rats modeled by bilateral common carotid artery occlusion (BCCAO), that aimed to improve the treatment methods for VD. 8-week-old healthy SPF male Wistar rats (280±20g) were randomly divided into the normal group (Gn, n=10), sham operated group (Gs, n=10), and operated group (Go, n=35). The VD rat models in Go group were established by BCCAO. 8weeks after surgery, the operated rats were screened by the hidden platform trail of Morris Water Maze (MWM), and the rats with cognitive dysfunction were further randomly divided into the impaired group (Gi, n=10) and TCM group (Gm, n=10). The VD rats in Gm group were given the intragastric administration of Huangdisan grain decoction once a day for 8weeks, and the other groups were given intragastric administration of normal saline. Then the cognitive ability of rats in each group was detected by the MWM Test. The lymphocyte subsets in peripheral blood and hippocampus of rats were measured by flow cytometry. The levels of cytokines (IL-1β, IL-2, IL-4, IL-10, TNF-α, INF-γ, MIP-2, COX-2, iNOS) in peripheral blood and hippocampus were measured by ELISA (enzyme linked immunosorbent assay). The number of Iba-1+ CD68+ co-positive cells in the CA1 region of hippocampus was measured by immunofluorescence. Compared with the Gn group, the escape latencies of the Gi group were prolonged (P<0.01), the time spent in the former platform quadrant was shortened (P<0.01), and the number of times of crossing over the former platform location was reduced (P<0.05). But compared with the Gi group, the escape latencies of Gm group were shortened (P<0.01), the time spent in the former platform quadrant was prolonged (P<0.05), and the number of times of crossing over the former platform location was increased (P<0.05). The number of Iba-1+ CD68+ co-positive cells in the CA1 region of hippocampus of VD rats in Gi group was increased (P<0.01) compared with the Gn group. And the proportions of T Cells, CD4+ T Cells, CD8+ T Cells in the hippocampus were increased (P<0.01). The level of pro-inflammatory cytokines in the hippocampus was increased significantly, such as IL-1β (P<0.01), IL-2 (P<0.01), TNF-α (P<0.05), IFN-γ (P<0.01), COX-2 (P<0.01), MIP-2 (P<0.01) and iNOS (P<0.05). And the level of IL-10 (P<0.01), a kind of anti-inflammatory cytokine, was decreased. The proportions of T Cells (P<0.05), CD4+ T Cells (P<0.01) and NK Cells (P<0.05) in the peripheral blood of the VD rats in Gi group were decreased, and the level of IL-1β, IL-2, TNF-α, IFN-γ, COX-2, MIP-2 and iNOS was increased significantly (P<0.01) compared with the Gn group. Meanwhile, the level of IL-4 and IL-10 was decreased (P<0.01). Huangdisan grain could reduce the number of Iba-1+ CD68+ co-positive cells in the CA1 region of hippocampus (P<0.01), decrease the proportions of T Cells, CD4+ T Cells, CD8+ T Cells and the level of IL-1β, MIP-2 in hippocampus (P<0.01) of VD rats. Moreover, it could rise the proportion of NK Cells (P<0.01) and the level of IL-4 (P<0.05), IL-10 (P<0.05), and decrease the level of IL-1β (P<0.01), IL-2 (P<0.05), TNF-α (P<0.01), IFN-γ (P<0.01), COX-2 (P<0.01) and MIP-2 (P<0.01) in peripheral blood of VD rats. This study indicated that Huangdisan grain could decrease the activation of microglia/macrophages, regulate the proportions of lymphocyte subsets and the level of cytokines, which could adjust the immunologic abnormalities of VD rats, and ultimately improve cognitive function.

Mechanism of resistance to phagocytosis and pulmonary persistence in mucoid Pseudomonas aeruginosa.

Pseudomonas aeruginosa is known for its ability to form biofilms, which are dependent on the production of exopolysaccharides. During chronic colonization of the airway and biofilm formation, P. aeruginosa converts to a mucoid phenotype, indicating production of the exopolysaccharide alginate. The mucoid phenotype promotes resistance to phagocytic killing, but the mechanism has not been established. To better understand the mechanism of phagocytic evasion conferred by alginate production, Human (THP-1) and murine (MH-S) macrophage cell lines were used to determine the effects of alginate production on macrophage binding, signaling and phagocytosis. Phagocytosis assays using mucoid clinical isolate FRD1 and its non-mucoid algD mutant showed that alginate production inhibited opsonic and non-opsonic phagocytosis, but exogenous alginate was not protective. Alginate caused a decrease in binding to murine macrophages. Blocking antibodies to CD11b and CD14 showed that these receptors were important for phagocytosis and were blocked by alginate. Furthermore, alginate production decreased the activation of signaling pathways required for phagocytosis. Mucoid and non-mucoid bacteria induced similar levels of MIP-2 from murine macrophages. This study demonstrated for the first time that alginate on the bacterial surface inhibits receptor-ligand interactions important for phagocytosis. Our data suggest that there is a selection for alginate conversion that blocks the earliest steps in phagocytosis, leading to persistence during chronic pulmonary infections.

Airway epithelial cell-specific deletion of HMGB1 exaggerates inflammatory responses in mice with muco-obstructive airway disease.

High mobility group box 1 (HMGB1), a ubiquitous chromatin-binding protein required for gene transcription regulation, is released into the extracellular microenvironment by various structural and immune cells, where it is known to act as an alarmin. Here, we investigated the role of airway epithelium-specific HMGB1 in the pathogenesis of muco-obstructive lung disease in Scnn1b-transgenic (Tg+) mouse, a model of human cystic fibrosis (CF)-like lung disease. We hypothesized that airway epithelium-derived HMGB1 modulates muco-inflammatory lung responses in the Tg+ mice. The airway epithelium-specific HMGB1-deficient mice were generated and the effects of HMGB1 deletion on immune cell recruitment, airway epithelial cell composition, mucous cell metaplasia, and bacterial clearance were determined. The airway epithelium-specific deletion of HMGB1 in wild-type (WT) mice did not result in any morphological alterations in the airway epithelium. The deficiency of HMGB1 in airway epithelial cells in the Tg+ mice, however, resulted in significantly increased infiltration of macrophages, neutrophils, and eosinophils which was associated with significantly higher levels of inflammatory mediators, including G-CSF, KC, MIP-2, MCP-1, MIP-1α, MIP-1β, IP-10, and TNF-α in the airspaces. Furthermore, as compared to the HMGB1-sufficient Tg+ mice, the airway epithelial cell-specific HMGB1-deficient Tg+ mice exhibited poor resolution of spontaneous bacterial infection. The HMGB1 deficiency in the airway epithelial cells of Tg+ mice did not alter airway epithelial cell-specific responses including epithelial cell proliferation, mucous cell metaplasia, and mucus obstruction. Collectively, our findings provide novel insights into the role of airway epithelial cell-derived HMGB1 in the pathogenesis of CF-like lung disease in Tg+ mice.

Expression profile of cytokines and chemokines in a mouse high-altitude cerebral edema model.

High-altitude cerebral edema (HACE) is considered to be the end-stage of acute mountain sickness (AMS); however, its pathophysiological mechanism remains unknown. Increasing evidences support that inflammation is an important risk factor for the occurrence of HACE. Including our published papers, previous studies demonstrated that the levels of IL-6, IL-1β, and TNF-α in both serum and hippocampus were increased in the mouse HACE model induced by LPS stimulation combined with hypobaric hypoxia exposure; however, the expression profile of other cytokines and chemokines remains unknown. This study was to analyze the expression profile of cytokines and chemokines in the HACE model. The mouse HACE model was established by LPS stimulation combined with hypobaric hypoxia exposure (LH). The mice were divided into the normoxic group, LH-6h group, LH-1d group, and LH-7d group. Brain water content (BWC) was determined using the wet/dry weight ratio. The levels of 30 cytokines and chemokines in the serum and hippocampal tissue were detected using LiquiChip. The mRNA expression of cytokines and chemokines in hippocampal tissue were determined by q-PCR. In the current study, we found that the brain water content was increased after the combinational treatment of LPS and hypobaric hypoxia. The results of LiquiChip showed that, in the serum and hippocampal tissue, most factors in all 30 cytokines and chemokines were dramatically upregulated at 6h, and then declined at the 1std and 7thd. Among these factors, G-CSF, M-CSF, MCP-1, KC, MIG, Eotaxin, Rantes, IP10, IL-6, MIP-2, and MIP-1β were all increased in both serum and hippocampal tissue at 6h. In addition, the results of q-PCR showed the mRNA levels of G-CSF, MCP-1, KC, MIG, Eotaxin, Rantes, IP10, IL-6, MIP-2, and MIP-1β in hippocampal tissue were dramatically upregulated at 6h. This study showed that the dynamic expression profile of 30 cytokines and chemokines in a mouse HACE model induced by LPS plus hypobaric hypoxia. The levels of G-CSF, MCP-1, KC, MIG, Eotaxin, Rantes, IP10, IL-6, MIP-2, and MIP-1β in both serum and hippocampus were significantly increased at 6h, which may be involved in the occurrence and development of HACE.

Cannabinoids inhibit ethanol-induced activation of liver toxicity in rats through JNK/ERK/MAPK signaling pathways.

Cannabinoids (CBs) are psychoactive compounds, with reported anticancer, anti-inflammatory, and anti-neoplastic properties. The study was aimed at assessing the hepatoprotective effects of CB against ethanol (EtOH)-induced liver toxicity in rats. The animals were divided into seven groups: control (Group I) and Group II were treated with 50% ethanol (EtOH 5 mg/kg). Groups III, IV, and VI were treated with (EtOH + CB 10 mg/kg), (EtOH + CB 20 mg/kg), and (EtOH + CB 30 mg/kg), respectively. Groups V and VII consisted of animals treated with 20 and 30 mg/kg, of CB, respectively. Biochemical analysis revealed that Group IV (EtOH + CB 20 mg/kg) had reduced levels of ALT-alanine transferase, AST-aspartate aminotransferase, ALP-alanine peroxidase, MDA-malondialdehyde and increased levels of GSH-reduced glutathione. Histopathological analysis of liver and kidney tissues showed that EtOH + CB (20 and 30 mg/kg) treated animal groups exhibited normal tissue architecture similar to that of the control group. ELISA revealed that the inflammatory markers were reduced in the animal groups that were treated with EtOH + CB 20 mg/kg, in comparison to the animals treated only with EtOH. The mRNA expression levels of COX-2, CD-14, and MIP-2 showed a remarkable decrease in EtOH + CB treated animal groups to control groups. Western blot analysis revealed that CB downregulated p38/JNK/ERK thereby exhibiting its hepatoprotective property by inhibiting mitogen-activated protein kinasepathways. Thus, our findings suggest that CB is a potential candidate for the treatment of alcohol-induced hepatotoxicity.

Conioselinum tenuissimum Root Extract Modulates Macrophage Activation via the Calcium–STAT3 Pathway

Despite the development of many antibiotics, excessive inflammation caused by endotoxins is still a subject of interest to biomedical researchers. The hyper-inflammatory response of macrophages activated by endotoxins is an important topic in the development of natural product-based anti-inflammatory drugs. Conioselinum tenuissimum, a perennial herb of the family Apiaceae, contains levistolide A, demethylsuberosin, and fraxetin. One of the synonyms of Conioselinum tenuissimum is Angelica tenuissima. The objective of this study was to determine the effects of Conioselinumtenuissimum root water extract (AT) on the hyper-inflammatory responses of macrophages activated by endotoxin (lipopolysaccharide; LPS) and the mechanisms involved in such effects. Levels of cytokines, nitric oxide (NO), hydrogen peroxide, and cytosolic calcium in LPS-activated RAW 264.7 murine macrophages were evaluated by the multiplex cytokine assay (MCA), Griess reagent assay (GRA), dihydrorhodamine 123 assay (DHR), and Fluo-4 calcium assay (FCA), respectively. Additionally, real-time PCR and the flow cytometry assay (FLA) was performed to determine the effects of AT on LPS-activated RAW 264.7. Data from MCA, GRA, DHR, and FCA revealed that AT lowered levels of IL-6, MCP-1, TNF-α, G-CSF, GM-CSF, VEGF, M-CSF, LIF, LIX, MIP-1α, MIP-1β, MIP-2, RANTES, IP-10, NO, hydrogen peroxide, and calcium in LPS-activated RAW 264.7. Real-time PCR results revealed that AT significantly lowered mRNA expression levels of inflammatory genes such as Chop, Nos2, c-Jun, Stat1, Stat3, c-Fos, Camk2a, Ptgs2, Fas, and Jak2. FLA showed that AT significantly reduced phosphorylation levels of P38 MAPK and STAT3 in LPS-activated RAW 264.7. These results indicate that AT can exert anti-inflammatory effects in LPS-activated macrophages via the calcium–STAT3 pathway.

Jinhua Qinggan granules attenuates acute lung injury by promotion of neutrophil apoptosis and inhibition of TLR4/MyD88/NF-κB pathway

Ethnopharmacological relevanceAcute lung injury (ALI) is one of the fatal complications of respiratory virus infections such as influenza virus and coronavirus, which has high clinical morbidity and mortality. Jinhua Qinggan granules (JHQG) has been approved by China Food and Drug Administration in the treatment of H1N1 influenza and mild or moderate novel coronavirus disease 2019 (COVID-19), which is an herbal formula developed based on Maxingshigan decoction and Yinqiao powder that have been used to respiratory diseases in China for thousands of years. However, the underlying mechanism of JHQG in treating infectious diseases remains unclear. Aim of the studyThis study investigated the effects of JHQG on neutrophil apoptosis and key signaling pathways in lipopolysaccharide (LPS) -induced ALI mice in order to explore its mechanism of anti-inflammation. Materials and methodsThe effect of JHQG on survival rate was observed in septic mouse model by intraperitoneal injection of LPS (20 mg/kg). To better pharmacological evaluation, the mice received an intratracheal injection of 5 mg/kg LPS. Lung histopathological changes, wet-to-dry ratio of the lungs, and MPO activity in the lungs and total protein concentration, total cells number, TNF-α, IL-1β, IL-6, and MIP-2 levels in BALF were assessed. Neutrophil apoptosis rate was detected by Ly6G-APC/Annexin V-FITC staining. Key proteins associated with apoptosis including caspase 3/7 activity, Bcl-xL and Mcl-1 were measured by flow cytometry and confocal microscope, respectively. TLR4 receptor and its downstream signaling were analyzed by Western blot assay and immunofluorescence, respectively. ResultsJHQG treatment at either 6 or 12 g/kg/day resulted in 20% increase of survival in 20 mg/kg LPS-induced mice. In the model of 5 mg/kg LPS-induced mice, JHQG obviously decreased the total protein concentration in BALF, wet-to-dry ratio of the lungs, and lung histological damage. It also attenuated the MPO activity and the proportion of Ly6G staining positive neutrophils in the lungs, as well as the MIP-2 levels in BALF were reduced. JHQG inhibited the expression of Mcl-1 and Bcl-xL and enhanced caspase-3/7 activity, indicating that JHQG partially acted in promoting neutrophil apoptosis via intrinsic mitochondrial apoptotic pathway. The levels of TNF-α, IL-1β, and IL-6 were significantly declined in LPS-induced mice treated with JHQG. Furthermore, JHQG reduced the protein expression of TLR4, MyD88, p-p65 and the proportion of nuclei p65, suggesting that JHQG treatment inhibited TLR4/MyD88/NF-κB pathway. ConclusionJHQG reduced pulmonary inflammation and protected mice from LPS-induced ALI by promoting neutrophil apoptosis and inhibition of TLR4/MyD88/NF-κB pathway, suggesting that JHQG may be a promising drug for treatment of ALI.

Local cytokine/chemokine profiles in BALB/c and C57BL/6 mice in response to T. vaginalis infection

Trichomonas vaginalis is the causative agent of Trichomoniasis (a sexually transmitted infection). Recent reports have shown that stimulation of cellular immunity can reduce trichomoniasis infection. Animal studies are essential to understanding the pathogenesis of infection and developing new potential drugs and vaccines to treat the infection. Therefore, we have tried to understand the pathogenesis of T. vaginalis infection by investigating the differences in the expression of chemokine/cytokine levels in vaginal and cervical tissues of BALB/c and C57BL/6 mice. Different pathological symptoms, like desquamation, neutrophil infiltration, and hemorrhage, were recorded in BALB/c and C57BL/6 in response to T. vaginalis infection. Vaginal and cervical tissues of BALB/c showed these symptoms on 2nd dpi, which became severe on 7th dpi and turned to mild or normal till 14th dpi compared to C57BL/6 strain. Immunohistochemistry in the vagina and cervical tissues of BALB/c and C57BL/6 mice was done to assess cytokines at different time intervals post-infection. Significant expression of Interleukin-1β (IL-1β) (a pro-inflammatory cytokine) was found in BALB/c compared to the C57BL/6 mice, on 7th dpi and 2nd dpi in vaginal and cervical tissues, respectively. Higher expression of MIP-2 (neutrophil chemoattractant) was observed in the vaginal tissues of BALB/c mice on 7th dpi compared to the C57BL/6 group. In addition, higher expression of TGF-β (immune-suppressor) was observed on 7th dpi in the vaginal tissue of BALB/c mice. The present study demonstrates that more pathological signs of T. vaginalis infection developed in BALB/c mice than C57BL/6 mice. Also, significant levels of IL-1β and MIP-2 were measured in BALB/c mice in response to T. vaginalis compared to C57BL/6.

TNF-α signaling is required for fungal clearance during brain infection with Cryptococcus neoformans via promoting the recruitment of CD4+ T cells and inflammatory monocytes

Abstract Cryptococcus neoformans is a pathogenic fungus accounting for 180,000 deaths worldwide per year. The infection starts in the lung; however, the fungus can migrate to the brain, causing fatal meningoencephalitis primarily in immunocompromised individuals. TNF-α has been shown to mediate protective immunity in the lung during pulmonary infection with C. neoformans. However, the functions of TNF-α signaling during brain infection with C. neoformans remain poorly understood. Recent studies have shown that CD4+ T cells and inflammatory monocytes (IMs) mediate cryptococcal clearance in the brain at the cost of inducing immunopathology. Here, we study the role of TNF-α in the context of CD4+ T cells and IMs during brain infection with C. neoformans. We found that TNF-α levels in the brain were significantly increased during infection. The increase of TNF-α was associated with enhanced mRNA expression of various chemokines and accumulation of CD4+ T cells and IMs. Genetic ablation of TNF-α receptor (TNF-αR) in mice led to significantly higher brain fungal burdens and early mortality during C. neoformans infection. Infected TNF-αR−/− mice displayed significantly reduced recruitment of CD4+ T cells and IMs in the brain compared to infected wild-type mice. This was accompanied with reduced mRNA levels of KC, MIP-2, CCL2 and CCL7. Interestingly, deficiency of TNF-αR also resulted in a significant drop in the mRNA levels of ICAM-1 and VCAM-1 as well as the expression of CD11a and VLA4 in CD4+ T cells and IMs. Collectively, these results suggest TNF-α signaling is essential for the recruitment of CD4+ T cells and IMs, mediating fungal clearance during brain infection with C. neoformans. Supported by AI131219 and AI131905

Experimental Arthritis Inhibits Adult Hippocampal Neurogenesis in Mice.

Background: Adult-born neurons of the hippocampal dentate gyrus play a role in specific forms of learning, and disturbed neurogenesis seems to contribute to the development of neuropsychiatric disorders, such as major depression. Neuroinflammation inhibits adult neurogenesis, but the effect of peripheral inflammation on this form of neuroplasticity is ambiguous. Objective: Our aim was to investigate the influence of acute and chronic experimental arthritis on adult hippocampal neurogenesis and to elucidate putative regulatory mechanisms. Methods: Arthritis was triggered by subcutaneous injection of complete Freund’s adjuvant (CFA) into the hind paws of adult male mice. The animals were killed either seven days (acute inflammation) or 21 days (chronic inflammation) after the CFA injection. Behavioral tests were used to demonstrate arthritis-related hypersensitivity to painful stimuli. We used in vivo bioluminescence imaging to verify local inflammation. The systemic inflammatory response was assessed by complete blood cell counts and by measurement of the cytokine/chemokine concentrations of TNF-α, IL-1α, IL-4, IL-6, IL-10, KC and MIP-2 in the inflamed hind limbs, peripheral blood and hippocampus to characterize the inflammatory responses in the periphery and in the brain. In the hippocampal dentate gyrus, the total number of newborn neurons was determined with quantitative immunohistochemistry visualizing BrdU- and doublecortin-positive cells. Microglial activation in the dentate gyrus was determined by quantifying the density of Iba1- and CD68-positive cells. Results: Both acute and chronic arthritis resulted in paw edema, mechanical and thermal hyperalgesia. We found phagocytic infiltration and increased levels of TNF-α, IL-4, IL-6, KC and MIP-2 in the inflamed hind paws. Circulating neutrophil granulocytes and IL-6 levels increased in the blood solely during the acute phase. In the dentate gyrus, chronic arthritis reduced the number of doublecortin-positive cells, and we found increased density of CD68-positive macrophages/microglia in both the acute and chronic phases. Cytokine levels, however, were not altered in the hippocampus. Conclusions: Our data suggest that acute peripheral inflammation initiates a cascade of molecular and cellular changes that eventually leads to reduced adult hippocampal neurogenesis, which was detectable only in the chronic inflammatory phase.

Comparison of serum cytokines for bacteremia and fungemia in rat bloodstream infection model

Objective We aim to measure the serum levels of cytokines/chemokines in cases of candidemia and bacteremia using mouse model. Methods and Materials: Mice models of bloodstream infection (BSI) with Staphylococcus aureus (Sa), Enterococcus faecalis (Ef), Escherichia coli (Ec), Klebsiella pneumonia (Kp), Candida albicans (Ca) and Candida parapsilosis (Cp) were established. Cytokines and chemokines including TNF-α, IL-1β, IL-10, IL-17, MIP-1β, MIP-2 and G-CSF in serum were detected by Luminex xMAP assay and were compared among different groups at different time points. Results: TNF-α, IL-1β and G-CSF first increased and then decreased after infection in all infection groups, while IL-10 decreased to the minimum levels 12h after injection and then gradually increased. As for IL-17, MIP-1β and MIP-2, they increased more rapidly than the other cytokines and then also decreased. The maximum levels of TNF-α, IL-1β, IL-17 MIP-1β and MIP-2 were all higher in GN group, when compared with GP groups and fungemia groups. Peak level of G-CSF in GP groups was higher than the other two groups, while IL-10 was the highest in fungemia groups. Conclusion: Our study indicates that variation trend of serum cytokines and chemokines were different in bacterial or fungal BSI models.

α- MSH Plays Anti-inflammatory and Anti-fungal Role in Aspergillus Fumigatus Keratitis

ABSTRACTPurposeTo investigate the anti-inflammatory and antifungal role of α-melanocyte stimulating hormone (α-MSH) in Aspergillus Fumigatus (A. fumigatus) keratitis.MethodCorneas of C57BL/6 mice were infected with A. Fumigatus. α-MSH (5 ul, 1×10−4 mmol/ml) was given by subconjunctival injection from day 1 to day 3 post infection (p.i.). After 3 days p.i., clinical score was recorded and HE staining was tested. Fungal load in mice corneas was observed by plate counting. Proinflammatory mediators and pattern recognition receptors (PRRs) were detected. The number of neutrophils and macrophages was tested by immunofluorescence staining. The role of α-MSH in RAW264.7 cells after A. fumigatus stimulation were evaluated by PCR and Western blot, and MPKA protein levels including total-JNK (T-JNK), phosphorylated-JNK (P-JNK), total-ERK (T-ERK), and phosphorylated-ERK (P-ERK) were tested via Western blot with or without α-MSH treatment.ResultsCompared with PBS control group, α-MSH treatment alleviated disease response and decreased clinical score at 3 days p.i. HE staining showed less infiltration in corneal tissue after α-MSH treatment. Plate counting experiment showed that number of viable fungus in corneas of α-MSH treated group was less than control group. mRNA levels of IL-1β, TNF-α, IL-6, MIP-2, LOX-1, Dectin-1, and iNOS were decreased. Protein levels of IL-1β, TNF-α, IL-6, and Dectin-1 were decreased. α-MSH treatment also decreased the infiltrating neutrophils and macrophages. The levels of proinflammatory cytokines, Dectin-1 and LOX-1 stimulated by A. fumigatus, were also suppressed by pretreatment of α-MSH in RAW264.7 cells. The ratio of P-JNK/T-JNK and P-ERK/T-ERK was downregulated in α-MSH group compared with PBS control group.Conclusionα-MSH alleviates the severity and decreases fungal load of A. fumigatus keratitis in mice. Migration of neutrophils and macrophages are restrained. α-MSH downregulates the expression of dectin-1 and the ratio of P-JNK/T-JNK and P-ERK/T-ERK in A. fumigatus infection.

Anakinra reduces lung inflammation in experimental acute lung injury.

IntroductionAcute respiratory distress syndrome (ARDS) is a severe form of acute lung injury (ALI) resulting in life‐threatening hypoxaemia. Although ARDS can be caused by a variety of pathogens or major trauma, it is best known as the major cause of mortality in COVID‐19 patients. Since ARDS is often associated with dysregulated inflammatory immune responses, immunomodulatory approaches represent a possible treatment option. The objective of this study was to evaluate the therapeutic potential of interleukin (IL)‐1 blockade using Anakinra in a mouse model of lipopolysaccharide (LPS)‐induced ALI.MethodsWe evaluated the effects of a daily subcutaneous Anakinra treatment in a mouse model of LPS‐induced ALI. We monitored body weight to assess the general health status of the mice. Two days after ALI induction, we evaluated the inflammatory cytokine MIP‐2 as well as protein levels in bronchoalveolar lavage (BAL) fluids. Two and nine days after ALI induction, we evaluated infiltrating leukocytes in BAL fluid and lung tissue.ResultsAnakinra treatment reduced ALI‐induced weight loss compared to nontreated groups. At Day 2, Anakinra treatment reduced levels of MIP‐2 and protein in BAL fluids and reduced frequencies of NK cells and neutrophils in the lung tissue. Nine days after ALI induction, Anakinra treated mice displayed reduced levels of neutrophils and alveolar macrophages in BAL fluids.ConclusionsIL‐1 blockade using Anakinra reduced classical hallmarks of inflammation in a mouse model of ALI. Our data support ongoing and future research on the evaluation of Anakinra as a potential treatment option in ARDS.