Lower Levels Of Proinflammatory Cytokines Research Articles

Biodegradable Nanoparticles Containing Antigen and Vitamin D3 Efficiently Induce Antigen-specific Immune Suppression

Abstract Biodegradable poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles (NPs) encapsulated with antigens can efficiently deliver the antigens to antigen-presenting cells such as dendritic cells (DCs). Active form of vitamin D, 1α,25-dihydroxyvitamin D3 (VD3), is known to have various immunosuppressive effects. Encapsulation of VD3 within NPs would enhance the delivery of VD3 to APCs, while preventing the potential systemic side effects of VD3. In this study, we examined the effects of PLGA NPs encapsulated with ovalbumin (OVA) and VD3. PLGA NPs containing OVA and VD3 (NP[OVA+VD3]) were prepared by W1/O/W2 double emulsion solvent evaporation method. Bone marrow-derived DCs treated with NP[OVA+VD3] were decreased in the expression of co-stimulatory molecules, and produced higher levels of immunosuppressive cytokines, while producing lower levels of pro-inflammatory cytokines, compared with DCs treated with NP[OVA]. Addition of NP[OVA+VD3] to allogeneic MLR reduced the proliferation of allogenic T cells. In addition, NP[OVA+VD3]-treated DCs efficiently generated CD4+CD25+Foxp3+Treg cells from CD4+CD25− T cells. Furthermore, mice injected i.v. with NP[OVA+VD3] were suppressed in inducing OVA-specific cytotoxic T cells. These results show that phagocytic delivery of antigen and VD3 to APCs using biodegradable NPs efficiently suppresses antigen-specific immune responses.

Pomalidomide Reduces Ischemic Brain Injury in Rodents

Stroke is a leading cause of death and severe disability worldwide. After cerebral ischemia, inflammation plays a central role in the development of permanent neurological damage. Reactive oxygen species (ROS) are involved in the mechanism of post-ischemic inflammation. The activation of several inflammatory enzymes produces ROS, which subsequently suppress mitochondrial activity, leading to further tissue damage. Pomalidomide (POM) is a clinically available immunomodulatory and anti-inflammatory agent. Prior cellular studies demonstrate that POM can mitigate oxidative stress and lower levels of pro-inflammatory cytokines, particularly TNF-α, which plays a prominent role in ischemic stroke-induced brain damage and functional deficits. To evaluate the potential value of POM in cerebral ischemia, POM was initially administered to transgenic mice chronically over-expressing TNF-α surfactant protein (SP)-C promoter (SP-C/TNF-α mice) to assess whether systemically administered drug could lower systemic TNF-α level. POM significantly lowered serum levels of TNF-α and IL-5. Pharmacokinetic studies were then undertaken in mice to evaluate brain POM levels following systemic drug administration. POM possessed a brain/plasma concentration ratio of 0.71. Finally, rats were subjected to transient middle cerebral artery occlusion (MCAo) for 60 min, and subsequently treated with POM 30 min thereafter to evaluate action on cerebral ischemia. POM reduced the cerebral infarct volume in MCAo-challenged rats and improved motor activity, as evaluated by the elevated body swing test. POM’s neuroprotective actions on ischemic injury represent a potential therapeutic approach for ischemic brain damage and related disorders, and warrant further evaluation.

Inhibition of glutamine metabolism accelerates resolution of acute lung injury.

Despite recent advances, acute respiratory distress syndrome (ARDS) remains a severe and often fatal disease for which there is no therapy able to reduce the underlying excessive lung inflammation or enhance resolution of injury. Metabolic programming plays a critical role in regulating inflammatory responses. Due to their high metabolic needs, neutrophils, macrophages, and lymphocytes rely upon glutamine metabolism to support activation and function. Additionally, during times of physiologic stress, nearly all cells, including fibroblasts and epithelial cells, require glutamine metabolism. We hypothesized that inhibiting glutamine metabolism reduces lung inflammation and promotes resolution of acute lung injury. Lung injury was induced by instilling lipopolysaccharide (LPS) intratracheally. To inhibit glutamine metabolism, we administered a glutamine analogue, 6‐diazo‐5‐oxo‐L‐norleucine (DON) that binds to glutamine‐utilizing enzymes and transporters, after injury was well established. Treatment with DON led to less lung injury, fewer lung neutrophils, lung inflammatory and interstitial macrophages, and lower levels of proinflammatory cytokines and chemokines at 5 and/or 7 days after injury. Additionally, DON led to earlier expression of the growth factor amphiregulin and more rapid recovery of LPS‐induced weight loss. Thus, DON reduced lung inflammation and promoted resolution of injury. These data contribute to our understanding of how glutamine metabolism regulates lung inflammation and repair, and identifies a novel target for future therapies for ARDS and other inflammatory lung diseases.

Polymeric Nanoparticles Containing Both Antigen and Vitamin D3 Induce Antigen-Specific Immune Suppression.

The active form of vitamin D3, 1,25-dihydroxyvitamin D3 (aVD3), is known to exert beneficial effects in the treatment of autoimmune diseases because of its immunosuppressive effects. However, clinical application of aVD3 remains limited because of the potential side effects, particularly hypercalcemia. Encapsulation of aVD3 within biodegradable nanoparticles (NPs) would enhance the delivery of aVD3 to antigen presenting cells, while preventing the potential systemic side effects of aVD3. In the present study, polymeric NPs containing ovalbumin (OVA) and aVD3 (NP[OVA+aVD3]) were prepared via the water-in-oil-in-water double emulsion solvent evaporation method, after which their immunomodulatory effects were examined. Bone marrow-derived immature dendritic cells (DCs) treated with NP(OVA+aVD3) did not mature into immunogenic DCs but were converted into tolerogenic DCs, which express low levels of co-stimulatory molecules and MHC class II molecules, produce lower levels of pro-inflammatory cytokines while increasing the production of IL-10 and TGF-β, and induce the generation of Tregs. Intravenous injection with NP(OVA+aVD3) markedly suppressed the generation of OVA-specific CTLs in mice. Furthermore, OVA-specific immune tolerance was induced in mice orally administered with NP(OVA+aVD3). These results show that biodegradable NPs encapsulating both antigen and aVD3 can effectively induce antigen-specific immune suppression.

Depletion and Reconstitution of Macrophages in Mice.

Macrophages are innate immune cells, which have important roles in the inflammatory response to infections or tissue injury, and have an equally important role in the resolution of inflammation. Macrophages play a key part in directing the innate immune response and subsequent adaptive immune response. They can acquire a variety of distinct but also overlapping activation states, depending on the local microenvironment, in order to perform these functions. Stimuli, such as IFNγ and LPS, can promote an inflammatory activation state, which is associated with the production of reactive oxygen species, and pro-inflammatory cytokines and chemokines. Immune complexes and LPS can promote an anti-inflammatory activation state to prevent damage to the host, which is associated with the production of high levels of the anti-inflammatory cytokine IL-10 and low levels of pro-inflammatory cytokines. Wound-healing macrophages can be activated by IL-4 or IL-13 and have roles in tissue remodeling and the resolution of inflammation. Macrophages are present in nearly every tissue of the body and are important for maintaining homeostasis, but their dysfunction can also lead to diseases, such as inflammatory bowel disease. To study the role macrophages play in a complex in vivo environment, depletion and reconstitution experiments can be utilized. Clodronate liposomes are an effective and versatile way to deplete macrophages in vivo; they can allow selective depletion from tissues of interest and can be used on transgenic mice. However, clodronate liposomes deplete all types of macrophages as well as dendritic cells, so other strategies are required in parallel to determine whether macrophages or macrophages of a particular activation state are required. Reconstitution of macrophages by adoptive transfer can be performed, with or without prior depletion, to further suggest that the observed effect is macrophage dependent. Macrophages activated ex vivo or macrophages from transgenic mice can be adoptively transferred during disease models to determine whether a specific protein or activation state affects disease outcome. Macrophage contribution to health and disease can be effectively studied using depletion with clodronate liposomes and by macrophage reconstitution, as demonstrated in this chapter.

Exposure to Antibiotics Affects Saponin Immersion-Induced Immune Stimulation and Shift in Microbial Composition in Zebrafish Larvae.

In the last decades, pollution of the environment by large scale use of antibiotics in agriculture and human medicine have led to increased antimicrobial resistance in both the environment and the host animal microbiome. Disturbances in the host microbiome can result in impaired immunity and reduced resilience of aquaculture species. Here, we investigated whether environmentally measured levels of the commonly used antibiotics ciprofloxacin and oxytetracycline influences the host microbiome and susceptibility toward saponin-induced immune stimulation in larval zebrafish. Firstly, neutrophil and macrophage reporter zebrafish larvae were exposed to different concentrations of soy saponin by immersion. A dose-dependent increase in neutrophil presence in the intestinal area was observed together with increased expression of immune genes il1b, tnfa, il22 and mmp9. To investigate the effect of antibiotics, larval zebrafish were immersed in ciprofloxacin or oxytetracycline in the presence or absence of a low dose of saponin. In vivo imaging revealed that antibiotic treatment did not reduce the number of neutrophils that were recruited to the intestinal area upon saponin exposure, although it did tend to lower pro-inflammatory cytokine levels. Microbial sequencing of whole larvae revealed that exposure to a low dose of saponin already shifted the microbial composition. The combination of oxytetracycline and saponin significantly increased α-diversity compared to the controls. In conclusion, the current study provides evidence that the combination of low levels of antibiotics with low levels of anti-nutritional factors (saponin) can induce inflammatory phenotypes and can modify the microbiota, which might lead to altered disease susceptibility.

In Vivo Safety and Regeneration of Long-Term Transported Amniotic Fluid Stem Cells for Renal Regeneration.

Despite major progress in stem cell therapy, our knowledge of the characteristics and tissue regeneration potency of long-term transported cells is insufficient. In a previous in vitro study, we established the optimal cell transport conditions for amniotic fluid stem cells (AFSCs). In the present study, the target tissue regeneration of long-term transported cells was validated in vivo. For renal regeneration, transported AFSCs were seeded on a poly(lactide-co-glycolide) scaffold and implanted in a partially resected kidney. The target tissue regeneration of the transported cells was compared with that of freshly harvested cells in terms of morphological reconstruction, histological microstructure reformation, immune cell infiltration, presence of induced cells, migration into remote organs, expression of inflammation/fibrosis/renal differentiation-related factors, and functional recovery. The kidney implanted with transported cells showed recovery of total kidney volume, regeneration of glomerular/renal tubules, low CD4/CD8 infiltration, and no occurrence of cancer during 40weeks of observation. The AFSCs gradually disappeared and did not migrate into the liver, lung, or spleen. We observed low expression levels of pro-inflammatory cytokines and fibrotic factors; enhanced expression of the genes Wnt4, Pax2, Wt1, and Emx2; and significantly reduced blood urea nitrogen and creatinine values. There were no statistical differences between the performance of freshly harvested cells and that of the transported cells. This study demonstrates that long-term transported cells under optimized conditions can be used for cell therapy without adverse effects on stem cell characteristics, in vivo safety, and tissue regeneration potency.

Commensal Escherichia coli Strains Can Promote Intestinal Inflammation via Differential Interleukin-6 Production.

Escherichia coli is a facultative anaerobic symbiont found widely among mammalian gastrointestinal tracts. Several human studies have reported increased commensal E. coli abundance in the intestine during inflammation; however, host immunological responses toward commensal E. coli during inflammation are not well-defined. Here, we show that colonization of gnotobiotic mice with different genotypes of commensal E. coli isolated from healthy conventional microbiota mice and representing distinct populations of E. coli elicited strain-specific disease phenotypes and immunopathological changes following treatment with the inflammatory stimulus, dextran sulfate sodium (DSS). Production of the inflammatory cytokines GM-CSF, IL-6, and IFN-γ was a hallmark of the severe inflammation induced by E. coli strains of Sequence Type 129 (ST129) and ST375 following DSS administration. In contrast, colonization with E. coli strains ST150 and ST468 caused mild intestinal inflammation and triggered only low levels of pro-inflammatory cytokines, a response indistinguishable from that of E. coli-free control mice treated with DSS. The disease development observed with ST129 and ST375 colonization was not directly associated with their abundance in the GI tract as their levels did not change throughout DSS treatment, and no major differences in bacterial burden in the gut were observed among the strains tested. Data mining and in vivo neutralization identified IL-6 as a key cytokine responsible for the observed differential disease severity. Collectively, our results show that the capacity to exacerbate acute intestinal inflammation is a strain-specific trait that can potentially be overcome by blocking the pro-inflammatory immune responses that mediate intestinal tissue damage.

Evaluation of Effect of Probiotics on Cytokine Levels in Critically Ill Children With Severe Sepsis: A Double-Blind, Placebo-Controlled Trial.

To evaluate the effect of probiotics on cytokines in children with severe sepsis. Randomized, double-blind, placebo-controlled trial. ICU of a tertiary care teaching hospital in North India. Children 3 months to 12 years old with severe sepsis. Enrolled children were randomized to probiotic (n = 50) and placebo (n = 50) groups. Probiotic group received VSL#3 (Danisco-Dupont USA, Madison, WI) (Lactobacillus paracasei, L. plantarum, L. acidophilus, L. delbrueckii, Bifidobacterium longum, B. infantis, B. breve, Streptococcus salivarius; maltose and silicon dioxide), and placebo group received maltose and silicon dioxide. Dose was 1 sachet twice daily for 7 days. Blood was collected on days 1 and 7 for estimation of interleukin-6, interleukin-12p70, interleukin-17, tumor necrosis factor-α, interleukin-10, and transforming growth factor -β1. "Primary outcome": Change in cytokine levels in probiotic and placebo groups from day 1 to 7. "Secondary outcomes": Sequential Organ Failure Assessment score, healthcare-associated infections, ICU stay, and mortality. On day 7, probiotic group had significantly lower levels of proinflammatory cytokines (interleukin-6 [80 vs 186 pg/mL, p = 0.001]; interleukin-12p70 [44 vs 79 pg/mL, p = 0.001]; interleukin-17 [217 vs 293 pg/mL, p = 0.01]; and tumor necrosis factor-α [192 vs 348 pg/mL, p = 0.01]) and higher levels of antiinflammatory cytokines (interleukin-10 [320 vs 240 pg/mL, p = 0.02] and transforming growth factor-β1 [311 vs 221 ng/mL, p = 0.01]) than placebo group. From day 1 to 7, probiotic group showed significant decrease in proinflammatory cytokines (interleukin-6 [196-80 pg/mL, p = 0.001]; interleukin-12p70 [71-44 pg/mL, p = 0.01]; interleukin-17 [258-217 pg/mL, p = 0.01]; and tumor necrosis factor-α [347-192 pg/mL, p = 0.001]) and increase in antiinflammatory cytokines (interleukin-10 [198-320 pg/mL, p = 0.001] and transforming growth factor-β1 [216-311 ng/mL, p = 0.001]) as compared to placebo group. Sequential Organ Failure Assessment score on day 7 was significantly less in probiotic group (1 vs 3). There was a nonsignificant trend toward lower incidence of healthcare-associated infections (14% vs 20%) and duration of ICU stay (6.5 vs 9 d) in probiotic group. Mortality was similar in two groups. Probiotics supplementation for 7 days resulted in significant decrease in proinflammatory and increase in antiinflammatory cytokines in children with severe sepsis.

Clinical and metabolic characterization of obese subjects without non-alcoholic fatty liver: A targeted metabolomics approach

IntroductionAs a small proportion of obese individuals do not develop metabolic complications and non-alcoholic fatty liver disease (NAFLD), this study aimed to provide a comprehensive clinical, metabolic and genetic description of obese subjects with healthy livers. MethodsA total of 183 subjects were stratified, according to BMI, presence of metabolic syndrome, biochemical liver tests and hepatic steatosis on ultrasound, into: (i) lean controls (n = 69); (ii) obese healthy (n = 50); and (iii)obese NAFLD (n = 62) groups.Detailed clinical, genetic and metabolic evaluations were then performed. ResultsObese healthy subjects did not differ in glucose parameters from lean controls, and had a lower rate of minor TM6SF2 gene variants compared with obese NAFLD (2/49 vs. 11/60, respectively; P = 0.035) and lean controls (13/64; P = 0.035), but significantly higher leptin concentrations than lean controls (P < 0.001); they also higher adiponectin concentrations (P < 0.001), and lower TNF-α and IL-6 concentrations (P = 0.01 and P < 0.001, respectively), than obese NAFLD subjects. Also, metabolomic studies identified ether- and ester-containing phospholipids [PC ae C44:6, PC ae C42:5, PC aa C40:4; P < 0.001, corrected by the false discovery rate (FDR) method] and found that the amino-acids lysine, glycine and isoleucine (FDR < 0.001) differed between the two obese groups, but not between lean controls and obese healthy subjects. ConclusionObese people with healthy livers are characterized by intact glucose homoeostasis, lower pro-inflammatory cytokine levels, and higher adiponectin and leptin concentrations compared with obese people with NAFLD. In addition, the major allele of TM6SF2, a set of phosphatidylcholines and several amino acids are associated with healthy livers in obesity.

Effects of Bifidobacterium probiotic on the treatment of chronic periodontitis: A randomized clinical trial.

AimThis randomized placebo‐controlled clinical trial evaluated the effect of Bifidobacterium animalis subsp. lactis (B. lactis) HN019‐containing probiotic lozenges as adjuvant to scaling and root planing (SRP) in patients with generalized chronic periodontitis.Materials and MethodsForty‐one chronic periodontitis patients were recruited and monitored clinically, immunologically, and microbiologically at baseline (before SRP) and 30 and 90 days after SRP. All patients were randomly assigned to a Test (SRP + Probiotic, n = 20) or Control (SRP + Placebo, n = 21) group. The probiotic lozenges were used twice a day for 30 days. The data were statistically analysed.ResultsThe Test group presented a decrease in probing pocket depth and a clinical attachment gain significantly higher than those of the Control group at 90 days. The Test group also demonstrated significantly fewer periodontal pathogens of red and orange complexes, as well as lower proinflammatory cytokine levels when compared to the Control group. Only the Test group showed an increase in the number of B. lactis HN019 DNA copies on subgingival biofilm at 30 and 90 days.ConclusionThe use of B. lactis HN019 as an adjunct to SRP promotes additional clinical, microbiological, and immunological benefits in the treatment of chronic periodontitis (NCT03408548).

An hPSC-Derived Tissue-Resident Macrophage Model Reveals Differential Responses of Macrophages to ZIKV and DENV Infection.

SummaryZika virus (ZIKV) and dengue virus (DENV) are two closely related flaviviruses that lead to different clinical outcomes. The mechanism for the distinct pathogenesis of ZIKV and DENV is poorly understood. Here, we investigate ZIKV and DENV infection of macrophages using a human pluripotent stem cell (hPSC)-derived macrophage model and discover key virus-specific responses. ZIKV and DENV productively infect hPSC-derived macrophages. DENV, but not ZIKV, infection of macrophages strongly activates macrophage migration inhibitory factor (MIF) secretion and decreases macrophage migration. Neutralization of MIF leads to improved migratory ability of DENV-infected macrophages. In contrast, ZIKV-infected macrophages exhibit prolonged migration and express low levels of pro-inflammatory cytokines and chemokines. Mechanistically, ZIKV disrupts the nuclear factor κB (NF-κB)-MIF positive feedback loop by inhibiting the NF-κB signaling pathway. Our results demonstrate the utility of hPSC-derived macrophages in infectious disease modeling and suggest that the distinct impact of ZIKV and DENV on macrophage immune response may underlie different pathogenesis of Zika and dengue diseases.

MTOR Inhibitors Maintain Low Levels of Immune Activation, Immune Senescence and EBV Load in Kidney Transplant Patients

Introduction Post-transplant lymphoproliferative disorders (PTLD) represent a severe complication in transplant patients. Epstein-Barr Virus (EBV) is the main driver of PTLD, particularly of those occurring early after transplantation. Immune activation/inflammation, immune senescence, reactivation of EBV and expansion of EBV-positive B cells, which play a pivotal role in the onset of PTLD, may be modulated by immunosuppressive strategies. The aim of this study was to assess the impact of mTOR inhibitor (mTORi) on viral and host cofactors involved in the PTLD’s pathogenesis. Materials and Methods Sixty-two kidney transplant patients were enrolled in the study: 33 were treated with mycophenolic acid (MPA) and 29 with mTORi, both in combination with tacrolimus and steroids. Thymoglobulin was the induction in all cases. All patients were assessed at the time of transplant and after 1 year of follow-up. Markers of T-cell activation(CD3+CD8+CD38+), exhaustion(CD3+CD8+PD-1+), senescence (CD3+CD8+CD28-CD57+), and B-cell activation (CD19+CD10-CD21lowCD27+) were evaluated by flow cytometry. Plasma levels of PAMPs(microbial translocation marker 16s ribosomal(r)DNA) and DAMPs(mitochondrial(mt)DNA) were quantified by real-time PCR. Inflammatory cytokines (IL-6 and TNF-alpha) were quantified by Luminex platform. EBV was typed and quantified by multiplex real-time PCR, and telomere lengths in peripheral blood cells (PBMC) were estimated by telomere/single copy gene ratio (T/S), using multiplex real-time PCR. Results After 1 year of follow-up, plasma levels of 16s rDNA increased in MPA-treated patients (p=0.029), while remained stable in the mTORi group. Levels of mtDNA and pro- inflammatory cytokines tended to decrease in mTORi-treated patients (mtDNA, p=0.034; IL-6, p=0.08; TNF-alpha, p<0.001), while no significant changes were observed in the MPA group. Percentages of activated, exhausted and senescent T cells significantly increased in the MPA-treated patients (4.6[2.2-9.1] vs 17.8[12.1-23.3],p<0.001; 4.8[3.0-6.9] vs 11.6[8.7-16.8],p<0.001; and 7.9[4.4-14.5] vs 14.5[8.5-27.7],p=0.005), but remained stable in the mTORi group (2.4[1.8-6.1] vs 4.1[2.4-6.3], p=0.161; 6.4[4.9-12.1] vs 7.1[4.3-12.7],p=0.888; and 9.7[5.2-13.8]vs 10.8[5.6-16.2],p=0.460). The increase in immunosenescent cells in the MPA group was supported by telomere shortening, higher in MPA than in mTORi-treated patients (ΔT/S=-0.449 vs ΔT/S=-0.219). Percentages of activated B cells and levels of EBV-DNA significantly increased in the MPA group (10.3[6.4-16.9] vs 18.3[11.6-28.4],p=0.002, and 1[1-17] vs 27[1-118] EBV-DNA copies/105 PBMC, p=0.004), while remained stable in the mTORi group (12.8[6.8-18.8]vs 13.0[9.2-18.0], p=0.836, and 1[1-7]vs 6[1-26] EBV-DNA copies/105 PBMC, p=0.128). Conclusions mTORi maintained low levels of pro-inflammatory cytokines, immune activation and senescence, thus constraining EBV reactivation and expansion of EBV-positive B cells. Overall, these findings suggest that mTORi may reduce the risk of EBV-related malignancies in kidney transplant patients. AIRC grant IG2016 Id 19112.

IL-37 isoform D downregulates pro-inflammatory cytokines expression in a Smad3-dependent manner

IL-37 is a new member of IL-1 family and possesses five different isoforms (named as IL-37 a–e). IL-37b has been demonstrated as a physiological suppressor of immune responses. However, the function of other isoforms remains unknown. Here, we show that IL-37d possesses anti-inflammatory roles both in vitro and in vivo. Firstly, IL-37d is expressed in peripheral blood mononuclear cells (PBMCs) and umbilical cords-derived mesenchymal stem cells (UCMSCs). Secondly, IL-37d overexpression markedly inhibits IL-1β-induced IL-6 production in A549 cells. Consistently, bone marrow-derived macrophages (BMDMs) from IL-37d transgenic mice express low levels of pro-inflammatory cytokines (such as IL-6 and TNF-α) following LPS stimulation, compared with those from wild-type mice. Furthermore, IL-37d transgenic mice produce less pro-inflammatory cytokines, and show much less degree of LPS-induced endotoxemia in vivo. Mechanistically, IL-37d interacts with Smad3 and promotes nuclear translocation of pSmad3. SIS3 (a specific Smad3 inhibitor) treatment completely blocks the inhibitory effects of IL-37d. Thus, our data indicate that IL-37d is a functional cytokine that negatively regulates pro-inflammatory cytokines expression in a Smad3-dependent manner.

M Protein of Group a Streptococcus Plays an Essential Role in Inducing High Expression of A20 in Macrophages Resulting in the Downregulation of Inflammatory Response in Lung Tissue.

Group A streptococcus (GAS), a common pathogen, is able to escape host immune attack and thus survive for longer periods of time. One of the mechanisms used by GAS is the upregulated expression of immunosuppressive molecules, which leads to a reduction in the production of inflammatory cytokines in immune cells. In the present study, we found that macrophages produced lower levels of proinflammatory cytokines (IL-1β, TNF-α, IL-6) when challenged with GAS than they did when challenged with Escherichia coli (E. coli). Simultaneously, in a mouse model of lung infection, GAS appeared to induce a weaker inflammatory response compared to E. coli. Our data also indicated that the expression of the A20 transcriptional regulator was higher in GAS-infected macrophages than that in macrophages infected with E. coli, and that high expression of A20 correlated with a reduction in the production of TRAF6. SiRNA targeting of A20 led to the increased production of TRAF6, IL-1β, TNF-α, and IL-6, suggesting that A20 inhibits synthesis of these key proinflammatory cytokines. We also investigated the pathway underlying A20 production and found that the synthesis of A20 depends on My88, and to a lower extent on TNFR1. Finally, we showed a significant reduction in the expression of A20 in macrophages stimulated by M protein-mutant GAS, however, a speB-GAS mutant, which is unable to degrade M protein, induced a greater level of A20 production than wild type GAS. Collectively, our data suggested that M protein of GAS was responsible for inducing A20 expression in macrophages, which in turn down-regulates the inflammatory cytokine response in order to facilitate GAS in evading immune surveillance and thus prolong survival in the host.

Loss of Sprouty2 enhances IL‐33 expression and protects against experimental colitis.

OBJECTIVESprouty2 (Spry2) is an intracellular modulator of MAPK signaling. It is present in the intestinal tract, but its function there is unknown. Aberrant MAPK signaling is associated with altered cytokine production in disorders such as inflammatory bowel disease, raising the possibility that Spry2 may affect the response to colitis. In particular, MAPK regulates IL‐33 expression in response to bacterial infection, and IL‐33 has been shown to be protective in some forms of colitis. Furthermore, both MAPK and IL‐33 regulate secretory cell differentiation. Here we tested the hypothesis that Spry2 control of MAPK signaling alters cytokine production and/or secretory cell development, thus altering the intestinal response to injury.METHODSMice that lack Spry2 in the intestinal epithelium (VillinCre;Spry2flox/flox, hereafter Spry2KOIE) and littermate controls were given 3% dextran sodium sulfate (DSS) in drinking water for 4 days followed by 3 days without DSS, and colitis severity was assessed. Cytokine levels and epithelial marker expression were determined by qPCR in challenged and unchallenged mice. GSK3beta phosphorylation was determined by western blot. In vitro, IEC6 and YAMC intestinal epithelial cells were treated with GSK3beta and MAPK inhibitors and cytokine expression was assessed.RESULTSColonic Spry2 levels were reduced in DSS colitis by 59% (p&lt;0.05). Spry2KOIE mice subjected to DSS had lower levels of pro‐inflammatory cytokines versus wildtype littermates, and were protected from histological damage (injury score reduced by 30%, p=0.037), weight loss, and colon shortening. Unchallenged Spry2KOIE mice displayed elevated GSK3beta phosphorylation on the inactivating Ser9 site in epithelial cells (88% increase, p&lt;0.01). These mice also had increased IL‐33 expression (80% increase, p&lt;0.05) and more DCLK1+ tuft cells in the colon (2.2‐fold increase, p&lt;0.01). In vitro, inactivation of GSK3beta in both IEC6 and YAMC cells mimicked the increased IL‐33 observed in vivo; this effect was attenuated by ERK or p38 MAPK inhibitors. Expression of the epithelial‐derived cytokine CXCL2 was unaffected by GSK3beta inhibition, suggesting an IL‐33 specific response.CONCLUSIONColonic epithelial Spry2 is downregulated in response to injury and inflammation. This may represent an integration point of environmentally‐driven MAPK signals that leads to altered Wnt signaling, elevated IL‐33 expression, and promotion of intestinal tuft cell development as a protective or regenerative mechanism.Support or Funding InformationNIH R01DK095004This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Fisetin-treatment alleviates airway inflammation through inhbition of MyD88/NF-κB signaling pathway.

Asthma is a common chronic airway inflammation disease and is considered as a major public health problem. Fisetin (3,3′,4′,7-tetrahydroxyflavone) is a naturally occurring flavonoid abundantly found in different vegetables and fruits. Fisetin has been reported to exhibit various positive biological effects, including anti-proliferative, anticancer, anti-oxidative and neuroprotective effects. We evaluated the effects of fisetin on allergic asthma regulation in mice. Mice were first sensi-tized, then airway-challenged with ovalbumin (OVA). Whether fisetin treatment attenuated OVA-induced airway inflammation was examined via inflammation inhibition through MyD88-related NF-κB (p65) signaling pathway. Mice were divided into the control (Con), OVA-induced asthma (Mod), 40 (FL) and 50 (FH) mg/kg fisetin-treated OVA-induced asthma groups. Our results found that OVA-induced airway inflammation in mice caused a significant inflammatory response via the activation of MyD88 and NF-κB signaling pathways, leading to release of pro-inflammatory cytokines. In contrast, fisetin-treated mice after OVA induction inhibited activation of MyD88 and NF-κB signaling pathways, resulting in downregulation of pro-inflammatory cytokine secretion. Further, fisetin significantly ameliorated the airway hyperresponsiveness (AHR) towards methacholine (Mch). In addition, fisetin reduced the number of eosinophil, monocyte, neutrophil and total white blood cell in the bronchoalveolar lavage fluid (BALF) of OVA-induced mice. The serum and BALF samples obtained from the OVA-induced mice with fisetin showed lower levels of pro-inflammatory cytokines. The results of our study illustrated that fisetin may be a new promising candidate to inhibit airway inflammation response induced by OVA.

Mesenchymal stromal cells of bone marrow and azathioprine in Crohn's disease therapy.

To evaluate the effectiveness of MSCs therapy in patients with CD receiving azathioprine (AZA). The study included 34 patients with inflammatory (luminal) form of CD. The 1st group of patients (n=15) received an- ti-inflammatory therapy using MSCs culture in combination with AZA. The 2nd group (n=19) received MSCs without AZA. The severity of the attack was assessed in points in accordance with the of Crohn's disease activity index (CDAI). Immunoglobulins (IgA, IgG, IgM), interleukins (IL) 1β, 4, 10, tumor necrosis factor-α (TNF-α), interferon-γ (INF-γ), transforming growth factor-1β (TGF-1β), C-reactive protein (CRP), platelets and erythrocyte sedimentation rate (ESR) at 2, 6 and 12 months from the beginning of MSCs therapy. The initial mean CDAI in the 1st group was 337.6±17.1 points, in the 2nd group - 332.7±11.0 points (p=0.3). In both groups of pa- tients there was a significant decrease in CDAI after 2 months. From the beginning of therapy MSCs: in the 1st group to 118.9±12.4 points, in the 2nd - 120.3±14.1 points (p=0.7), after 6 months - 110.3±11.1 and 114.3±11.8 points (p=0.8), respectively. After 12 months CDAI in the 1st group was 99.9±10.8 points, in the 2nd group it was 100.6±12.1 points (p=0.8). The level of IgA, IgG, IgM was significantly lower in the group of patients with a longer history of the disease and long-term ASA. After the introduction of MSC in both groups of patients with BC, there was a tendency for the growth of pro- and anti-inflammatory cytokines, with a significantly lower level of pro-inflammatory cytokines - INF-γ, TNF-α, IL-1β - in the 1st group, indicating potentiation of the immunosuppressive effect of MSCs and AZA, which provides a more pro- nounced anti-inflammatory effect. Transplantation of MSCs promotes an increase in the serum of patients with CD initially reduced concentration of IG, cytokines and restoring their balance as the onset of clinical remission. The combination with AZA has a more pronounced anti-inflammatory effect.

Tumour vasculature immaturity, oxidative damage and systemic inflammation stratify survival of colorectal cancer patients on bevacizumab treatment.

Despite treatment of patients with metastatic colorectal cancer (mCRC) with bevacizumab plus chemotherapy, response rates are modest and there are no biomarkers available that will predict response. The aim of this study was to assess if markers associated with three interconnected cancer-associated biological processes, specifically angiogenesis, inflammation and oxidative damage, could stratify the survival outcome of this cohort.Levels of angiogenesis, inflammation and oxidative damage markers were assessed in pre-bevacizumab resected tumour and serum samples of mCRC patients by dual immunofluorescence, immunohistochemistry and ELISA.This study identified that specific markers of angiogenesis, inflammation and oxidative damage stratify survival of patients on this anti-angiogenic treatment. Biomarkers of immature tumour vasculature (% IMM, p=0.026, n=80), high levels of oxidative damage in the tumour epithelium (intensity of 8-oxo-dG in nuclear and cytoplasmic compartments, p=0.042 and 0.038 respectively, n=75) and lower systemic pro-inflammatory cytokines (IL6 and IL8, p=0.053 and 0.049 respectively, n=61) significantly stratify with median overall survival (OS).In summary, screening for a panel of biomarkers for high levels of immature tumour vasculature, high levels of oxidative DNA damage and low levels of systemic pro-inflammatory cytokines may be beneficial in predicting enhanced survival outcome following bevacizumab treatment for mCRC.

Effects of low‐dose epinephrine on perioperative hemostasis and inflammatory reaction in major surgical operations: a randomized clinical trial

Background Hemostasis, thrombosis and surgical stress-induced immune reactions are important for perioperative morbidity and recovery after major surgical operations. Objectives To evaluate the effects of combined administration of low-dose epinephrine (LDEPI) and tranexamic acid (TXA) on perioperative blood loss, thromboembolic complications and inflammatory responses in patients undergoing total hip arthroplasty (THA). Patients/Methods Patients scheduled for THA (n = 195) were randomized into three interventions: intravenous LDEPI plus TXA (group IV); topical diluted epinephrine plus TXA (group TP); and TXA alone as control (group CT). The primary outcome was perioperative blood loss on postoperative day (POD) 1. Secondary outcomes included perioperative blood loss on POD 3, intraoperative blood loss, volume of drainage, transfusion values, coagulation and fibrinolysis parameters, inflammatory cytokine levels, cases of thrombosis, intravenous fluid on the operation day, and length of hospital stay. Results The mean calculated amounts of total blood loss in groups IV, TP and CT were 631.2 mL, 760.5 mL, and 825.6 mL, respectively, on POD 1; treatment effects (differences) were 194.4 mL (95% confidence interval [CI] 146.7-242.0) and 65.0 mL (95% CI 17.4-112.7). Groups IV and TP had lower levels of proinflammatory cytokines (tumor necrosis factor-α and interleukin [IL]-1β) and higher levels of the anti-inflammatory cytokine IL-10, and showed faster development of coagulation and fibrinolysis (without change in peak levels), than group CT early postoperation. No differences were observed in transfusion, thromboembolic and other outcomes among the groups. Conclusion The combined administration of LDEPI and TXA was more effective in reducing perioperative blood loss and alleviating the inflammatory response than TXA alone, without increasing the incidence of thromboembolic and other complications.