Reduced TNF-α Production Research Articles

Oreocharioside A-G, new acylated C-glycosylflavones from Oreocharis auricula (Gesneriaceae)

Seven new acylated C-glycosylflavones, oreocharioside A-G, together with two known compounds were isolated from the whole plant of Oreocharis auricula. Their structures were characterized by the comprehensive analysis of their NMR, IR, UV, CD spectra and HRESIMS data. All the new compounds were evaluated for the antioxidant and anti-inflammatory activities. The results showed that compounds 1 and 2 had significant DPPH and ABTS radical scavenging activities, with the IC50 values of 0.32–3.20 μg/mL. Compounds 2 and 3 exhibited the higher potency among all the new compounds in reducing TNF-α production.

Polysucrose hydrogel and nanofiber scaffolds for skin tissue regeneration: Architecture and cell response

Scaffolds capable of mediating overlapping multi-cellular activities to support the different phases of wound healing while preventing scarring are essential for tissue regeneration. The potential of polysucrose as hydrogels and electrospun mats for wound healing was evaluated in vitro by seeding fibroblasts, endothelial cells and macrophages either singly or in combination. It was found that the scaffold architecture impacted cell behaviour. Electrospun mats promoted fibroblasts flattened morphology while polysucrose methacrylate (PSucMA) hydrogels promoted fibroblast spheroids formation, accentuated in the presence of endothelial cells. Hydrogels exhibited lower inflammatory response than mats and curcumin loaded scaffolds reduced TNF-α production. In vivo biocompatibility of the hydrogels tested on Wistar rats was superior to electrospun mats. In vivo wound healing studies indicated that PSucMA hydrogels integrated the surrounding tissue with better cellular infiltration and proliferation throughout the entire wound region. PSucMA hydrogels led to scarless wound closure comparable with commercially available gels.

Hydrolyzed low-molecular-weight polysaccharide from Enteromorpha prolifera exhibits high anti-inflammatory activity and promotes wound healing

Increased anti-inflammatory activity is key to accelerating wound healing. In this study, we prepared three types of water-soluble polysaccharides from Enteromorpha prolifera (PEPs) with different molecular weights by hydrochloric acid hydrolysis and evaluated their anti-inflammatory activity and wound-healing effects. The results showed that, under in vitro conditions, the low molecular-weight polysaccharide (LPEP) exhibited high anti-inflammatory activity and suppressed the production of nitric oxide (NO), tumour necrosis factor α (TNF-α), and interleukins (IL-1β, and IL-6) from macrophage RAW 264.7 cells through the TLR2-dependent NF-κB, PKC/ERK/MAPK, and PI3K/Akt signalling cascades, in a dose-dependent manner. Under in vivo conditions, the LPEP showed remarkable anti-inflammatory activity, effectively reduced TNF-α production within 14 days, and significantly promoted wound healing to reach wound closure rates of up to 99.0%. Further, a higher number of fibroblast cells and a lower number of inflammatory cells were observed around the wound site in LPEP- and amoxicillin-treated rats than in 0.9% NaCl-treated rats. Similarly, faster tissue regeneration and more skin appendages were found for these two treatment groups. This study demonstrates the potential of LPEP as an effective anti-inflammatory agent to reduce inflammation and promote wound healing.

The dual effect of acetate on microglial TNF-α production

IntroductionShort-Chain Fatty Acids (SCFA) are products of intestinal microbial metabolism that can reach the brain and alter microglia in health and disease contexts. However, data are conflicting on the effect of acetate, the most abundant SCFA in the blood, in these cells. ObjectiveThe authors aimed to investigate acetate as a modulator of the inflammatory response in microglia stimulated with LPS. MethodThe authors used an immortalized cell line, C8-B4, and primary cells for in vitro treatments with acetate and LPS. Cell viability was analyzed by MTT, cytokine by RT-PCR, ELISA, and flow cytometry. The authors also performed in vivo and in silico analyses to study the role of acetate and the TNF-α contribution to the development of Experimental Autoimmune Encephalomyelitis (EAE). ResultsAcetate co-administered with LPS was able to exacerbate the production of pro-inflammatory cytokines at gene and protein levels in cell lines and primary culture of microglia. However, the same effects were not observed when acetate was administered alone or as pretreatment, prior to the LPS stimulus. Additionally, pharmacological inhibition of histone deacetylase concomitantly with acetate and LPS led to decreased TNF-α production. In silico analysis showed a crucial role of the TNF-α pathway in EAE development. Moreover, acetate administration in vivo during the initial phase of EAE led to a better disease outcome and reduced TNF-α production. ConclusionTreatment with acetate was able to promote the production of TNF-α in a concomitant LPS stimulus of microglia. However, the immune modulation of microglia by acetate pretreatment may be a component in the generation of future therapies for neurodegenerative diseases.

Iguratimod decreases bleomycin-induced pulmonary fibrosis in association with inhibition of TNF-α in mice

Severe interstitial lung disease secondary to connective tissue diseases, characterized by pulmonary inflammation and fibrosis, often have very poor prognosis due to lack of effective treatments. Iguratimod (IGU) shows encouraging efficacy in treating connective tissue diseases, however, the underlying mechanism is still to be elucidated. In this study, we investigated the impact of IGU on bleomycin-induced interstitial lung disease and the related tumor necrosis factor-α (TNF-α) signaling pathway in mice and in the alveolar epithelial cell A549. We found IGU decreased pulmonary inflammation and fibrosis and expression of fibrosis-related genes such as Collagen I, α-smooth muscle actin (α-SMA) and matrix metalloproteinase-2 (MMP-2) induced by bleomycin. IGU inhibited epithelial-mesenchymal transition as evidenced by decreased E-cadherin expression but increased vimentin expression. IGU reduced TNF-α production in the pulmonary fibrosis murine model and in the in vitro cultured A549 cells. Furthermore, IGU ameliorated TNF-α-induced severe pulmonary fibrosis and inhibited TNF-α-induced activation of NF-κB. In addition, IGU decreased IL-6 production and phosphorylation of STAT3. In conclusion, the IGU-mediated anti-fibrogenesis effect was associated with the inhibition of TNF-α and NF-κB.

Intratumor Regulatory Noncytotoxic NK Cells in Patients with Hepatocellular Carcinoma.

Previous studies support the role of natural killer (NK) cells in controlling hepatocellular carcinoma (HCC) progression. However, ambiguity remains about the multiplicity and the role of different NK cell subsets, as a pro-oncogenic function has been suggested. We performed phenotypic and functional characterization of NK cells infiltrating HCC, with the corresponding nontumorous tissue and liver from patients undergoing liver resection for colorectal liver metastasis used as controls. We identified a reduced number of NK cells in tumors with higher frequency of CD56BRIGHTCD16− NK cells associated with higher expression of NKG2A, NKp44, and NKp30 and downregulation of NKG2D. Liver-resident (CXCR6+) NK cells were reduced in the tumors where T-bethiEomeslo expression was predominant. HCCs showed higher expression of CD49a with particular enrichment in CD49a+Eomes+ NK cells, a subset typically represented in the decidua and playing a proangiogenic function. Functional analysis showed reduced TNF-α production along with impaired cytotoxic capacity that was inversely related to CXCR6−, T-bethiEomeslo, and CD49a+Eomes+ NK cells. In conclusion, we identified a subset of NK cells infiltrating HCC, including non-liver-resident cells that coexpressed CD49a and Eomes and showed reduced cytotoxic potential. This NK cell subset likely plays a regulatory role in proangiogenic function.

Solidagenone acts on promastigotes of L. amazonensis by inducing apoptosis-like processes on intracellular amastigotes by IL-12p70/ROS/NO pathway activation

BackgroundLeishmaniasis is a neglected tropical disease caused by protozoan parasites of the Leishmania genus. Currently, the treatment has limited effectiveness and high toxicity, is expensive, requires long-term treatment, induces significant side effects, and promotes drug resistance. Thus, new therapeutic strategies must be developed to find alternative compounds with high efficiency and low cost. Solidagenone (SOL), one of the main constituents of Solidago chilensis, has shown gastroprotective, anti-inflammatory and immunomodulatory effects. PurposeThis study assessed the in vitro effect of SOL on promastigotes and Leishmania amazonensis-infected macrophages, as well its microbicide and immunomodulatory mechanisms. MethodsSOL was isolated from the roots of S. chilensis, 98% purity, and identified by chromatographic methods, and the effect of SOL on leishmanicidal activity against promastigotes in vitro, SOL-induced cytotoxicity in THP-1, J774 cells, sheep erythrocytes, and L. amazonensis-infected J774 macrophages, and the mechanisms of death involved in this action were evaluated. ResultsIn silico predictions showed good drug-likeness potential for SOL with high oral bioavailability and intestinal absorption. SOL treatment (10–160 μM) inhibited promastigote proliferation 24, 48, and 72 h after treatment. After 24 h of treatment, SOL at the IC50 (34.5 μM) and 2 × the IC50 (69 μM) induced several morphological and ultrastructural changes in promastigotes, altered the cell cycle and cellular volume, increased phosphatidylserine exposure on the cell surface, induced the loss of plasma membrane integrity, increased the reactive oxygen species (ROS) level, induced loss of mitochondrial integrity (characterized by an apoptosis-like process), and increased the number of lipid droplets and autophagic vacuoles. Additionally, SOL induced low cytotoxicity in J774 murine macrophages (CC50 of 1587 μM), THP-1 human monocytes (CC50 of 1321 μM), and sheep erythrocytes. SOL treatment reduced the percentage of L. amazonensis-infected macrophages and the number of amastigotes per macrophage (IC50 9.5 μM), reduced TNF-α production and increased IL-12p70, ROS and nitric oxide (NO) levels. ConclusionSOL showed in vitro leishmanicidal effects against the promastigotes by apoptosis-like mechanism and amastigotes by reducing TNF-α and increasing IL-12p70, ROS, and NO levels, suggesting their potential as a candidate for use in further studies on the design of antileishmanial drugs.

Vitamin D3 Controls TLR4- and TLR2-Mediated Inflammatory Responses of Endometrial Cells

Objectives: Vitamin D has potent immunoregulatory features and modulates innate and adaptive immune responses. There is a significant association between intrauterine infection-associated inflammatory responses and pregnancy complications such as abortion and preterm labor. Here, we investigated how 1,25 (OH)2 D3 could modulate inflammatory responses of endometrial cells. Design: This is an in vitro experimental study. Endometrial stromal cells (ESCs) and whole endometrial cells (WECs) were collected from 15 apparently normal women, and the immunomodulatory effects of 1,25 (OH)2 D3 on lipopolysaccharide (LPS)- or lipoteichoic acid (LTA)-treated ESCs and WECs were investigated. Participants/Materials, Setting, and Methods: Women with no history of abortion, infertility, endometriosis, or sign of vaginal infection were enrolled in this study. Endometrial samples were collected by gynecologists using a Pipelle pipette in the proliferative phase of the menstrual cycle. WECs and ESCs were collected and treated with either LPS or LTA. The levels of IL-6, IL-8, and TNF-α in culture supernatants were quantified using the ELISA technique. TLR2, TLR4, and MyD88 expressions were assessed by RT-qPCR. TLR4 expression at the protein level was studied by the Western blot technique. Results: 1,25 Dihydroxycholecalciferol (1,25 (OH)2 D3) significantly reduced TNF-α production in LPS-activated ESCs and TNF-α and IL-6 production by LTA-stimulated WECs. In contrast, 1,25 (OH)2 D3 pretreatment increased the production of IL-8 by LPS- and LTA-stimulated endometrial cells. 1,25 (OH)2 D3 pretreatment markedly reduced LPS-induced TLR4 protein expression by ESCs. LPS treatment of ESCs significantly induced MyD88 gene expression. This effect was reversed when these cells were pretreated with 1,25 (OH)2 D3 before stimulation with LPS. Limitations: Because of the small size of samples, doing experiments all together on some samples was not feasible. Confirmation of the results obtained here needs well-designed in vivo studies. Conclusions: 1,25 (OH)2 D3 is an immunomodulatory molecule essential for maintaining endometrial immune homeostasis by controlling potentially harmful inflammatory responses associated with female reproductive tract infections.

Dilodendron bipinnatum Radlk. extract alleviates ulcerative colitis induced by TNBS in rats by reducing inflammatory cell infiltration, TNF-α and IL-1β concentrations, IL-17 and COX-2 expressions, supporting mucus production and promotes an antioxidant effect

Ethnopharmacological relevanceDilodendron bipinnatum (Sapindaceae) stem bark decoction and macerate were used to treat uterine inflammation, pain in general, dermatitis and bone fractures. These homemade preparations also have diuretic, stimulant, expectorants and sedative effects and are effective in treating worm infections in the Brazilian Pantanal population. Our previous research confirmed the anti-inflammatory activity of the hydroethanolic extract of inner stem bark of D. bipinnatum (HEDb). AimThis work aimed to investigate the efficacy of HEDb in ameliorating experimental colitis in rats and to elucidate the possible mechanisms involved in the anti-ulcerative colitis properties of HEDb in rats and Caco-2 cell line. Materials and methodsThe effects on cell viability, IL-8 and TNF-α in human colon adenocarcinoma (Caco-2) were determined by flow cytometer and ELISA. Wistar rats (n = 6–7) were orally gavaged with, vehicle (0.9% saline), HEDb at doses of 20, 100 or 500 mg/kg, or mesalazine at a dose of 500 mg/kg, at 48, 24 and 1 h prior to the administration of trinitrobenzene sulfonic acid via rectal administration to induce colitis. The anti-inflammatory effects of HEDb were assessed macroscopically, by myeloperoxidase (MPO) activity and for glutathione (GSH) concentration in the colon. Additionally, colonic histopathological analyses of UC severity were conducted by different staining methods (H&E, PAS and toluidine blue). Pro-inflammatory cytokines TNF-α and IL-1β were quantified in colonic tissue by ELISA and colonic expressions of COX-2 and IL-17 were analyzed by western blotting. ResultsHEDb was shown to be non-cytotoxic with mean viability of 80% in Caco-2 cells. HEDb pre-treatments of 1, 5 or 20 μg/mL significantly reduced TNF-α production in Caco-2 cells by 21.8% (p < 0.05), 60.5 and 82.1% (p < 0.001) respectively following LPS treatment compared to LPS alone. However, no change in IL-8 production was observed. HEDb pre-treatment of rats subjected to TNBS significantly (p < 0.001) reduced colonic lesion score. Higher doses (100 and 500 mg/kg) caused a sharp downregulation of haemorrhagic damage, leukocyte infiltration, edema and restoration of mucus production. Moreover, mast cell degranulation was inhibited. Colonic MPO activity was reduced following all doses of HEDb, reaching 51.1% ± 1.51 (p < 0.05) with the highest dose. GSH concentration was restored by 58% and 70% following 100 and 500 mg/kg of HEDb, respectively. The oral treatment of HEDb at doses 20, 100 and 500 mg/kg decreased the concentrations of TNF-α and IL-1β at all doses in comparison to vehicle treated control. In addition, HEDb inhibited the COX-2 and IL-17 expressions with maximal effect at 500 mg/kg (60.3% and 65% respectively; p < 0.001). In all trials, the effect of HEDb at all doses being 20, 100 and 500 mg/kg was statistically comparable to mesalazine (500 mg/kg). ConclusionsHEDb reduces colonic damage in the TNBS colitis model and relieves oxidative and inflammatory events, at least in part, by increasing mucus production, reducing leukocyte migration and reducing TNF-α (in vivo and in vitro), IL-1β, IL-17 and COX-2 expression. Therefore, HEDb requires further investigation as a candidate for treating IBD.

Splenic erythroid progenitors decrease TNF-α production by macrophages and reduce systemic inflammation in a mouse model of T cell-induced colitis.

In inflammatory bowel disease (IBD), inflammation can occur beyond the intestine and spread systemically causing complications such as arthritis, cachexia, and anemia. Here, we determine the impact of CD45, a pan-leukocyte marker and tyrosine phosphatase, on IBD. Using a mouse model of T cell transfer colitis, CD25- CD45RBhigh CD4+ T cells were transferred into Rag1-deficient mice (RAGKO) and CD45-deficient RAGKO mice (CD45RAGKO). Weight loss and systemic wasting syndrome were delayed in CD45RAGKO mice compared to RAGKO mice, despite equivalent inflammation in the colon. CD45RAGKO mice had reduced serum levels of TNF-α, and reduced TNF-α production by splenic myeloid cells. CD45RAGKO mice also had increased numbers of erythroid progenitors in the spleen, which had previously been shown to be immunosuppressive. Adoptive transfer of these erythroid progenitors into RAGKO mice reduced their weight loss and TNF-α expression by splenic red pulp macrophages. In vitro, erythroid cells suppressed TNF-α expression in red pulp macrophages in a phagocytosis-dependent manner. These findings show a novel role for erythroid progenitors in suppressing the pro-inflammatory function of splenic macrophages and cachexia associated with IBD.

PSIII-38 Butyric acid and derivatives: In vitro anti-inflammatory effects tested in porcine alveolar macrophages

Abstract The aim of this experiment was to examine the anti-inflammatory effects of butyric acid, sodium butyrate, monobutyrin and tributyrin using porcine alveolar macrophages (PAMs). PAMs were isolated from the bronchial lavage of 6 piglets at 6 weeks of age, and then seeded at 106 cells/mL in 24-well plates. After 24 h incubation, cells were treated with different treatments in a randomized complete block design with 10 replicates. The treatments were in a factorial arrangement with 2 doses of lipopolysaccharide (LPS, 0 or 1 μg/mL) and 5 levels of organic acid (0, 0.5, 1, 2, 4 mM for butyric acid and tributyrin and 0, 1, 2, 4, 8 mM for sodium butyrate and monobutyrin). Supernatants were collected after another 24 h incubation and analyzed for tumor necrosis factor alpha (TNF-α). Cell viability was also tested by the MTT assay. Data were analyzed using the MIXED procedure of SAS. No cytotoxic effect was observed in LPS challenge and each organic acid with the percentage of live cells was more than 76% in comparison to the sham control. Sodium butyrate at 2 and 4 mM dose exhibited (P &amp;lt; 0.01) a stimulatory effect on cell proliferation. LPS challenge remarkably stimulated (P &amp;lt; 0.0001) TNF-α secretion from PAMs. In the non-challenge group, butyric acid, monobutyrin, and tributyrin linearly reduced TNF-α production from PAMs, whereas 2 mM sodium butyrate tended to increase (P = 0.056) TNF-α secretion from PAMs. In the LPS challenge group, all tested organic acid dose-dependently reduced (P &amp;lt; 0.001) TNF-α production from LPS-challenged PAMs, with the strongest inhibiting effect observed at the highest dose. Results indicated that butyric acid and its derivatives that were tested in the current experiment all had strong anti-inflammatory activities in vitro.

Hydrogen sulphide ameliorates dopamine-induced astrocytic inflammation and neurodegeneration in minimal hepatic encephalopathy.

It has been demonstrated that the action of dopamine (DA) could enhance the production of tumour necrosis factor‐α (TNF‐α) by astrocytes and potentiate neuronal apoptosis in minimal hepatic encephalopathy (MHE). Recently, sodium hydrosulfide (NaHS) has been found to have neuroprotective properties. Our study addressed whether NaHS could rescue DA‐challenged inflammation and apoptosis in neurons to ameliorate memory impairment in MHE rats and in the neuron and astrocyte coculture system. We found that NaHS suppressed DA‐induced p65 acetylation, resulting in reduced TNF‐α production in astrocytes both in vitro and in vivo. Furthermore, decreased apoptosis was observed in neurons exposed to conditioned medium from DA + NaHS‐challenged astrocytes, which was similar to the results obtained in the neurons exposed to TNF‐α + NaHS, suggesting a therapeutic effect of NaHS on the suppression of neuronal apoptosis via the reduction of TNF‐α level. DA triggered the inactivation of p70 S6 ribosomal kinase (S6K1) and dephosphorylation of Bad, resulting in the disaggregation of Bclxl and Bak and the release of cytochrome c (Cyt. c), and this process could be reversed by NaHS administration. Our work demonstrated that NaHS attenuated DA‐induced astrocytic TNF‐α release and ameliorated inflammation‐induced neuronal apoptosis in MHE. Further research into this approach may uncover future potential therapeutic strategies for MHE.

Chlorogenic Acid Potentiates the Anti-Inflammatory Activity of Curcumin in LPS-Stimulated THP-1 Cells.

The anti-inflammatory effects of curcumin are well documented. However, the bioavailability of curcumin is a major barrier to its biological efficacy. Low-dose combination of complimentary bioactives appears to be an attractive strategy for limiting barriers to efficacy of bioactive compounds. In this study, the anti-inflammatory potential of curcumin in combination with chlorogenic acid (CGA), was investigated using human THP-1 macrophages stimulated with lipopolysaccharide (LPS). Curcumin alone suppressed TNF-α production in a dose-dependent manner with a decrease in cell viability at higher doses. Although treatment with CGA alone had no effect on TNF-α production, it however enhanced cell viability and co-administration with curcumin at a 1:1 ratio caused a synergistic reduction in TNF-α production with no impact on cell viability. Furthermore, an qRT-PCR analysis of NF-κB pathway components and inflammatory biomarkers indicated that CGA alone was not effective in reducing the mRNA expression of any of the tested inflammatory marker genes, except TLR-4. However, co-administration of CGA with curcumin, potentiated the anti-inflammatory effects of curcumin. Curcumin and CGA together reduced the mRNA expression of pro-inflammatory cytokines [TNF-α (~88%) and IL-6 (~99%)], and COX-2 (~92%), possibly by suppression of NF-κB (~78%), IκB-β-kinase (~60%) and TLR-4 receptor (~72%) at the mRNA level. Overall, co-administration with CGA improved the inflammation-lowering effects of curcumin in THP-1 cells.

Low back pain in athletes can be controlled with acupuncture by a catecholaminergic pathway: clinical trial.

Activation of the sympathetic nervous system attenuates inflammation via catecholamines. Recent evidence has shown that electroacupuncture (EA) activates neuronal networks involved in the release of dopamine and norepinephrine that control systemic inflammation. In muscle, catecholamines are related to cyclic adenosine monophosphate (cAMP). This signaling molecule has been implicated in recovery from sustained contractile activity, which may induce muscular pain, such as that which occurs during low back pain (LBP). Our aim was to evaluate the effects of EA used for the control of LBP on the activation of the sympathetic nervous system in a randomized controlled clinical trial in athletes. Two groups of athletes with acute or chronic low back pain were studied. EA, sham EA and pharmacological treatment (diclofenac sodium) were evaluated. The outcome measures included a pain score represented by a visual analogue scale (VAS) and serum levels of catecholamines quantified by enzyme-linked immunosorbent assay. In addition, blood was collected into chilled heparin tubes, placed in 96-well cell culture plates and incubated with an equal volume of Roswell Park Memorial Institute (RPMI) medium, with lipopolysaccharide (LPS) alone or with catecholamines. Tumor necrosis factor (TNF)-α levels in the supernatants were analyzed. The results indicated that the initial pain ratings did not differ between the groups analyzed. EA induced epinephrine secretion but not norepinephrine or dopamine secretion. Although EA and pharmacological treatment did not differ in terms of pain relief, in vitro epinephrine and norepinephrine reduced TNF-α production in response to LPS stimuli. EA activates the sympathetic nervous system and induces the release of epinephrine, which could ameliorate inflammation and protect muscular tissue in addition to relieving pain.

Alteration in TNFα mediates impaired antigen-specific humoral responses in sepsis survivors

Abstract Sepsis survivors show impaired responsiveness to antigen (Ag), which is linked to increased susceptibility to infection. Using the cecal ligation and puncture (CLP) model of sepsis, we showed that CLP surviving mice have a reduced antibody response to the T-dependent Ag, NP-CGG, compared to mice that underwent a sham procedure, but an intact response to the T-independent Ag, NP-Ficoll. CLP mice have reduced TNFα levels due to sustained vagus nerve activity. We hypothesized that reduced TNFα production might lead to disruption in follicular dendritic cell (FDC) function and impaired germinal center (GC) responses. FDCs play a key role in GC responses and TNFα is critical for FDC clustering and maturation. Immunofluorescence staining in spleens showed a reduced number of FDC clusters and reduced binding of immune complexes on FDCs in CLP mice compared to sham mice. NP-specific GC B cells sorted from CLP mice immunized with NP-CGG exhibit reduced TNFα, AICDA, and BCL6 mRNA expression compared to sham mice. To confirm the role of the vagus nerve in impaired FDC clusters, CLP mice were subjected to bilateral subdiaphragmatic vagotomy 2 weeks post-CLP. These mice exhibit increased anti-NP IgG levels in response to immunization with NP-CGG compared to non-vagotomized CLP mice. FDC are radioresistant cells; therefore, we lethally irradiated sham and CLP mice and reconstituted them with bone marrow cells from naive mice that never experienced sepsis. CLP mice still show an impaired Ag-specific response confirming involvement of FDCs and vagus nerve signals. In summary, our data suggest that altered vagus nerve activity, low TNFα, and disruption in FDC function contributes to reduced Ag-specific humoral responses in sepsis survivors.

Modulating inflammatory macrophages with an apoptotic body-inspired nanoparticle

Macrophages play a critical role in the initiation, maintenance, and resolution of inflammation because of their diverse and plastic phenotypic responses to extracellular stimuli. Inflammatory stimuli drive the recruitment and activation of inflammatory (M1) macrophages, capable of significant cytokine production that potentiates inflammation. Local environmental signals including apoptotic cell efferocytosis drive a phenotypic transition toward pro-reparative (M2) macrophages to facilitate the resolution of inflammation. However, prolonged or dysregulated inflammatory macrophage response contributes to many disease states and tissue damage. We have developed a nanoparticle to help resolve macrophage-mediated inflammation by mimicking the anti-inflammatory effect of apoptotic cell engulfment. The nanoparticle, comprised of a poly(lactide-co-glycolide) core, is coated in phosphatidylserine (PS)-supplemented cell plasma membrane to emulate key characteristics of the apoptotic cell surface. These apoptotic body-inspired PS/membrane-coated nanoparticles (PS-MNPs) reduce inflammatory cytokine expression to promote an anti-inflammatory, phenotypic shift in macrophages in vitro, without the use of small molecule inhibitors or other drugs. Specifically, PS-MNP treatment before lipopolysaccharide (LPS)-induced inflammatory challenge resulted in a 2.5-fold reduction in secreted tumor necrosis factor α (TNFα) at 24 h, with co-treatment of PS-MNPs and LPS demonstrating a 5-fold TNFα reduction compared to LPS alone. Reduced TNFα production, as well as gene expression of several pro-inflammatory cytokines, correlated with a reduction in NFκB activation from PS-MNP treatment. The development of a nanoparticle to reduce the production of multiple inflammatory cytokines and transition away from an inflammatory macrophage phenotype, through the use of a physiologic anti-inflammatory pathway, illustrates a new potential strategy in creating anti-inflammatory therapeutics. Statement of SignificanceMacrophages propagate inflammation as the major source of cytokine production in the body. In inflammatory diseases, pro-inflammatory macrophages persist in the site of inflammation and exacerbate tissue destruction. Current anti-inflammatory drugs have significant drawbacks, including variable response rates and off-target effects. Here, we have developed an apoptotic-body inspired nanoparticle to modulate inflammatory macrophage phenotype. This polymeric nanoparticle is coated with phosphatidylserine-supplemented cell plasma membrane to mimic the anti-inflammatory effect of apoptotic cell engulfment. Nanoparticle delivery reduces inflammatory cytokine production and promotes an anti-inflammatory phenotypic macrophage shift. The capacity of these nanoparticles to help resolve macrophage-mediated inflammation may be a useful tool to study macrophage-apoptotic cell interactions, the role of macrophages in inflammatory diseases, and in the design of anti-inflammatory therapeutics.

Extracellular CIRP induces macrophage endotoxin tolerance through IL-6R-mediated STAT3 activation.

Extracellular cold-inducible RNA-binding protein (eCIRP) is a damage-associated molecular pattern, whose effect on macrophages is not entirely elucidated. Here we identified that eCIRP promotes macrophage endotoxin tolerance. Septic mice had higher serum levels of eCIRP; this was associated with a reduced ex vivo immune response of their splenocytes to LPS. Pretreatment of macrophages with recombinant murine CIRP (rmCIRP) resulted in a tolerance to LPS stimulation as demonstrated by a reduction of TNF-α production. We found that eCIRP increased phosphorylated STAT3 (p-STAT3) in macrophages. A STAT3 inhibitor, Stattic, rescued macrophages from rmCIRP-induced tolerance by restoring the release of TNF-α in response to LPS stimulation. We discovered strong binding affinity between eCIRP and IL-6 receptor (IL-6R) as revealed by Biacore, fluorescence resonance energy transfer (FRET), and their colocalization in macrophages by immunostaining assays. Blockade of IL-6R with its neutralizing Ab inhibited eCIRP-induced p-STAT3 and restored LPS-stimulated TNF-α release in macrophages. Incubation of macrophages with rmCIRP skewed them toward an M2 phenotype, while treatment with anti–IL-6R Ab prevented rmCIRP-induced M2 polarization. Thus, we have demonstrated that eCIRP activates p-STAT3 via a novel receptor, IL-6R, to promote macrophage endotoxin tolerance. Targeting eCIRP appears to be a new therapeutic option to correct immune tolerance in sepsis.

An Anti-Inflammatory Composition of Boswellia serrata Resin Extracts Alleviates Pain and Protects Cartilage in Monoiodoacetate-Induced Osteoarthritis in Rats.

The boswellic acids, the active compounds in Boswellia serrata gum resin extract, are potent anti-inflammatory agents and are specific nonredox inhibitors of 5-Lipoxygenase (5-LOX). Here, we present the anti-osteoarthritis (OA) efficacy of LI13019F1 (also known as Serratrin®), a unique composition containing the acidic and nonacidic fractions of B. serrata gum resin. This composition strongly inhibited 5-LOX activity with the half-maximal inhibitory concentration (IC50) of 43.35 ± 4.90 μg/mL. Also, LI13019F1 strongly inhibited the leukotriene B4 (IC50, 7.80 ± 2.40 μg/mL) and prostaglandin E2 (IC50, 6.19 ± 0.52 μg/mL) productions in human blood-derived cells. Besides, LI13019F1 reduced TNF-α production with the IC50 of 12.38 ± 0.423 μg/mL. On average, 1, 2.5, and 5 μg/mL doses of LI13019F1 protected 34.62, 47.66, and 62.29% SW1353 human chondrosarcoma cells from IL-1β induced SOX-9 depletion, respectively. Further, a 28-day preclinical proof-of-concept study evaluated the pain relief efficacy of LI13019F1 in monoiodoacetate- (MIA-) induced Sprague-Dawley rats. At the end of the study, 150 and 300 mg/kg doses of LI13019F1 supplemented rats showed significant improvements (55.17 ± 5.81 g (p < 0.05), and 66.22 ± 6.30 g (p < 0.05), respectively, vs. MIA: 31.22 ± 7.15 g) in body-weight-bearing capacities. Concurrently, LI13019F1-150 and LI13019F1-300 rats substantially (p < 0.05) increased the threshold of pain sensitivity to pressure (26.98 ± 2.36 and 28.06 ± 2.72-gram force, respectively; vs. 18.63 ± 5.82 in MIA) and increased (p < 0.05) the latent time to withdraw the paw after a thermal stimulus (23.61 ± 2.73 and 28.18 ± 1.90 sec, respectively; vs. 16.56 ± 1.22 sec. in MIA). Besides, the histological observations on Safranin-O green stained articular cartilage revealed that LI13019F1 also prevented the MIA-induced structural damage of the cartilage and reduced the loss of the extracellular matrix (ECM) components in the experimental rats. In conclusion, the present observations suggest that LI13019F1, a new composition of B. serrata gum resin extracts, reduces pain and protects articular cartilage from the damaging action of MIA in a rodent model.

The modulatory effects of the PDE4 inhibitors CHF6001 and roflumilast in alveolar macrophages and lung tissue from COPD patients

BackgroundWe compared the anti-inflammatory effects of phosphodiesterase type 4 (PDE4) inhibitor roflumilast with CHF6001, a novel PDE4 inhibitor designed for inhaled administration, using human alveolar macrophages (AM) and lung tissue explants models. MethodsAM from 13 chronic obstructive pulmonary disease (COPD) patients and 10 smoking controls and lung tissue from 7 COPD patients were stimulated with LPS following preincubation with roflumilast (0.000001–10 µM), CHF6001 (0.000001–0.1 µM), or vehicle. After 24 h, supernatants were analysed for cytokines by ELISA. The effects of both compounds on the phosphorylation and cellular localisation of cAMP response element binding protein (CREB) were assessed by immunofluorescence and Western blot analysis. Extracted RNA was used for quantitative PCR analysis of PDE4 A, B and D mRNA. ResultsPDE4 A, B and D expression were increased in alveolar macrophages and lung tissue of COPD patients compared to controls. Roflumilast and CHF6001 significantly reduced TNF-α production in AM and lung tissue. CHF6001 was more potent than roflumilast with lower EC50s of 0.02, 0.01 and 0.31 nM compared to 0.87, 0.47 and 10.8 nM in respective samples. PDE4 inhibition also inhibited secretion of the chemokines CCL2 and CCL4 from macrophages. Both compounds increased nuclear levels of phosphorylated CREB. ConclusionPDE4 inhibitors caused a robust anti-inflammatory effect on TNF-α production from COPD AM, with inhibition of selective chemokines also observed. CHF6001 caused more potent inhibition of TNF-α production from COPD AM and lung tissue compared to roflumilast.

In vitro effects of poly(ADP-ribose) polymerase inhibitors on the production of tumor necrosis factor-α by interferon- γ - and lipopolysaccharide-stimulated peripheral blood mononuclear cells of horses.

To evaluate effects of poly(ADP-ribose) polymerase-1 (PARP1) inhibitors on the production of tumor necrosis factor-α (TNF-α) by interferon-γ (IFN-γ)- and lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells (PBMCs) of horses as an in vitro model of inflammation in horses. 1,440 samples of PBMCs from 6 healthy research horses. From heparinized whole blood samples, PBMC cultures were obtained. An initial dose-response trial on 48 PBMC samples from 2 horses (24 samples each) was used to determine concentrations of IFN-γ and LPS for use as low- and high-level stimulation concentrations. Seventy-two PBMC samples from 6 horses were assigned equally to 1 of 4 PARP1 inhibition categories: no PARP1 inhibitor (PARP1 inhibition control); 2-((R)-2-methylpyrrolidin-2-yl)-1H-benzimidazole-4-carbozamide dihydrochloride (ABT888);4-(3-(1-(cyclopropanecarbonyl)piperazine-4-carbonyl)-4-fluorobenzyl)phthalazin-1(2H)-one (AZD2281); or N-(6-oxo-5,6-dihydrophenanthridin-2-yl) -N,N-dimethylacetamide hydrochloride (PJ34). Samples of PBMCs from each horse and each PARP1 inhibition category were then assigned to 1 of 3 levels of IFN-γ and LPS stimulation: none (control), low stimulation, or high stimulation. After a 24-hour incubation period, a TNF-α ELISA was used to measure TNF-α concentration in the supernatant. Results were compared across treatments and for each horse. Data were analyzed with repeated-measures ANOVA. Median TNF-α concentration was significantly lower for PJ34-treated, high-level stimulated PBMCs than for PARP1 inhibition control, high-level stimulated PBMCs; however, no other meaningful differences in TNF-α concentration were detected among the inhibition and stimulation combinations. Findings suggested that PJ34 PARP1 inhibition may reduce TNF-α production in horses, a potential benefit in reducing inflammation and endotoxin-induced damage in horses.