Cytokine Expression In Response Research Articles

Low-molecular weight branched polyethylenimine reduces cytokine secretion from human immune system monocytes stimulated with bacterial and fungal PAMPs.

Adaptive immunity recruits T-cells and specific antibodies against antigens, innate immune cells express pathogen recognition receptors (PRRs) that can detect various pathogen-associated molecular patterns (PAMPs) released by invading pathogens. Microbial molecular patterns, such as lipopolysaccharide (LPS) from Gram-negative bacteria, trigger signaling cascades in the host that result in the production of pro-inflammatory cytokines. LPS stimulation produces a strong immune response and excessive LPS signaling leads to dysregulation of the immune response. However, dysregulated inflammatory response during wound healing often results in chronic non-healing wounds that are difficult to control. In this work, we present data demonstrating partial neutralization of anionic LPS molecules using cationic branched polyethylenimine (BPEI). The anionic sites on the LPS molecules from Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae) are the lipid A moiety and BPEI binding create steric factors that hinder the binding of PRR signaling co-factors. This reduces the production of pro-inflammatory TNF-α cytokines. However, the anionic sites of Pseudomonas aeruginosa (P. aeruginosa) LPS are in the O-antigen region and subsequent BPEI binding slightly reduces TNF-α cytokine production. Fortunately, BPEI can reduce TNF-α cytokine expression in response to stimulation by intact P. aeruginosa bacterial cells and fungal zymosan PAMPs.

IL-33 Induces Cellular and Exosomal miR-146a Expression as a Feedback Inhibitor of Mast Cell Function.

IL-33 is an inflammatory cytokine that promotes allergic disease by activating group 2 innate lymphoid cells, Th2 cells, and mast cells. IL-33 is increased in asthmatics, and its blockade suppresses asthma-like inflammation in mouse models. Homeostatic control of IL-33 signaling is poorly understood. Because the IL-33 receptor, ST2, acts via cascades used by the TLR family, similar feedback mechanisms may exist. MicroRNA (miR)-146a is induced by LPS-mediated TLR4 signaling and serves as a feedback inhibitor. Therefore, we explored whether miR-146a has a role in IL-33 signaling. IL-33 induced cellular and exosomal miR-146a expression in mouse bone marrow-derived mast cells (BMMCs). BMMCs transfected with a miR-146a antagonist or derived from miR-146a knockout mice showed enhanced cytokine expression in response to IL-33, suggesting that miR-146a is a negative regulator of IL-33-ST2 signaling. In vivo, miR-146a expression in plasma exosomes was elevated after i.p. injection of IL-33 in wild-type but not mast cell-deficient KitW-sh/W-sh mice. Finally, KitW-sh/W-sh mice acutely reconstituted with miR-146a knockout BMMCs prior to IL-33 challenge had elevated plasma IL-6 levels compared with littermates receiving wild-type BMMCs. These results support the hypothesis that miR-146a is a feedback regulator of IL-33-mediated mast cell functions associated with allergic disease.

HPV vaccines induce trained immunity and modulate pro-inflammatory cytokine expression in response to secondary Toll-like receptor stimulations.

Cervical cancer is caused mostly by human papillomavirus (HPV), and several HPV vaccines have been developed to prevent its onset. Vaccines include antigens as well as adjuvants, with adjuvants playing an important role in activating the innate immune responses necessary for inducing adaptive immunological responses. Recent research has shown the presence of trained immunity inside the innate immune system. However, trained immunity conferred by HPV vaccinations is not well understood. In this work, we explored the innate immune responses and trained immunity caused by two HPV vaccines, Cervarix and Gardasil. Cervarix includes monophosphoryl lipid A and an aluminum adjuvant, and it significantly increased the expression of IL-6 and IFN-β mRNAs in RAW264.7 cells. On the contrary, Gardasil, which only includes an aluminum adjuvant, exhibited little cytokine expression but increased the expression of TLRs. Furthermore, Cervarix significantly increased IL-1β secretion from mouse macrophages, while Gardasil only mildly induced IL-1β secretion. Interestingly, initial stimulation with Gardasil enhanced the expression of IL-6 and TNF-α mRNAs upon secondary stimulation with TLR ligands, indicating that Gardasil induced trained immunity in macrophages. Moreover, Gardasil injection into mice resulted in enhanced TNF-α production in sera following secondary TLR stimulation. Our findings suggest that HPV vaccinations have the ability to induce trained immunity that modulate TLR ligand responses.

Creeping fat exhibits distinct Inflammation-specific adipogenic preadipocytes in Crohn's disease.

Creeping fat (CrF) is an extraintestinal manifestation observed in patients with Crohn's disease (CD). It is characterized by the accumulation of mesenteric adipose tissue (MAT) that wraps around the intestinal wall. Although the role of CrF in CD is still debated, multiple studies have highlighted a correlation between CrF and inflammation, as well as fibrostenosais of the intestine, which contributes to the worsening of CD symptoms. However, the mechanism underlying the potential role of CrF in the development of Crohn's fibrosis remains an enigma. This study aimed to analyze CrF comprehensively using single-cell RNA sequencing analysis. The data was compared with transcriptomic data from adipose tissue in other disease conditions, such as ulcerative colitis, lymphedema, and obesity. Our analysis classified two lineages of preadipocyte (PAC) clusters responsible for adipogenesis and fibrosis in CrF. Committed PACs in CrF showed increased cytokine expression in response to bacterial stimuli, potentially worsening inflammation in patients with CD. We also observed an increase in fibrotic activity in PAC clusters in CrF. Co-analyzing the data from patients with lymphedema, we found that pro-fibrotic PACs featured upregulated pentraxin-3 expression, suggesting a potential target for the treatment of fibrosis in CrF. Furthermore, PACs in CrF exhibited a distinct increase in cell-to-cell communication via cytokines related to inflammation and fibrosis, such as CCL, LIGHT, PDGF, MIF, and SEMA3. Interestingly, these interactions also increased in PACs of the lymphedema, whereas the increased MIF signal of PACs was found to be a distinct characteristic of CrF. In immune cell clusters in CrF, we observed high immune activity of pro-inflammatory macrophages, antigen-presenting macrophages, B cells, and IgG+ plasma cells. Finally, we have demonstrated elevated IgG+ plasma cell infiltration and increased pentraxin-3 protein levels in the fibrotic regions of CrF in CD patients when compared to MAT from both UC patients and healthy individuals. These findings provide new insights into the transcriptomic features related to the inflammation of cells in CrF and suggest potential targets for attenuating fibrosis in CD.

Hyperthermic Enhancement of Immunotherapy: Findings of In Vitro Modeling

Despite advancement in understanding and manipulation of immune checkpoint molecules in immunotherapeutic design, limitations in treatment efficacy persist. Strategies to enhance effectiveness include use of multiple immunotherapeutic agents or combination with radiation therapy. Prior studies have also shown potential for hyperthermia to augment response to both of these therapeutic modalities. We hypothesized that in vitro assessment of moderate hyperthermia effects on the anti-tumor immune response will aid in development of targeted strategies that best combine hyperthermia with other immune manipulating therapies. To understand the consequences of temperature on carcinogenic phenotypes in vitro, B16-F10 melanoma cells were grown at 37°C or 41°C and biochemical profiles including protein expression were evaluated. Impact of hyperthermia on cell migration and proliferation were also assessed as were changes in the immune milieu including cytokine expression in response to heat. Data obtained was used to define ongoing in vivo experiments in which B16-F10 cells are implanted into C57BL/6 mice, grown to palpable tumors than treated with infrared radiation in combination with either anti-PDL1, anti-PD-1, or IL-15. Future studies based on these initial in vivo studies will explore integration of radiotherapy with hyperthermia and immunotherapy. B16-F10 cells grown at 41°C decreased cell migration by 70% in 24 hours, and decreased proliferation by 62% at 48 hours and 94% at 72 hours. To assess biochemical orchestrations exemplified by these data, protein expression profiles were evaluated. Expression of pERK and ERK decreased by 86% and 50% and caspase-3 increased by 31% at 41°C. Activation of sphingomyelinase and caspase-3 both rely on caspase-8. Sphingomyelinase activation results in CD95 receptor translocation, leading to cell death initiation in melanoma cells. Cell stress can induce death pathways and the heat shock protein response simultaneously. Of note, Hsp70 has an established role in fostering a tumor specific immune response. Thus, we investigated inducible hsp70 expression. Hsp70 expression increased by 188% at 41°C vs. 37°C. To evaluate the immune milieu, cytokine array data from conditioned media showed that at 41°C, TNFa expression was increased and IL-4 expression was decreased, suggesting a proinflammatory shift in cytokine profiles at hyperthermic temperatures. In support of our data, hyperthermia-induced TNFa apoptotic responses have been reported. In direct relation to clinical practice, we observed that hyperthermic potentiation decreased PDL1 expression in B16-F10 by 35%. Our work to date supports the hypothesis that hyperthermia can enhance immunotherapy via several mechanisms. In vivo study of the ability of hyperthermia to augment immune modulating therapies such as checkpoint blockade and radiation therapy is warranted.

Comparative effects of CFTR modulators on phagocytic, metabolic and inflammatory profiles of CF and nonCF macrophages

Macrophage dysfunction has been well-described in Cystic Fibrosis (CF) and may contribute to bacterial persistence in the lung. Whether CF macrophage dysfunction is related directly to Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) in macrophages or an indirect consequence of chronic inflammation and mucostasis is a subject of ongoing debate. CFTR modulators that restore CFTR function in epithelial cells improve global CF monocyte inflammatory responses but their direct effects on macrophages are less well understood. To address this knowledge gap, we measured phagocytosis, metabolism, and cytokine expression in response to a classical CF pathogen, Pseudomonas aeruginosa in monocyte-derived macrophages (MDM) isolated from CF F508del homozygous subjects and nonCF controls. Unexpectedly, we found that CFTR modulators enhanced phagocytosis in both CF and nonCF cohorts. CFTR triple modulators also inhibited MDM mitochondrial function, consistent with MDM activation. In contrast to studies in humans where CFTR modulators decreased serum inflammatory cytokine levels, modulators did not alter cytokine secretion in our system. Our studies therefore suggest modulator induced metabolic effects may promote bacterial clearance in both CF and nonCF monocyte-derived macrophages.

Contribution of peripheral blood mononuclear cells isolated by advanced filtration system to myogenesis of human bone marrow mesenchymal stem cells co-cultured with myoblasts

BackgroundContribution of peripheral blood mononuclear cells (PBMCs) in myogenesis is still under debate, even though blood filtration systems are commonly used in clinical practice for successfully management of critic limb ischemia. ObjectivesA commercial blood filter used for autologous human PBMC transplantation procedures is characterized and used to collect PBMCs, that are then added to well-established 2D in vitro myogenic models assembled with a co-culture of human bone marrow-derived mesenchymal stem cells (hBM-MSCs) and skeletal myoblasts (hSkMs) whit the aim of investigating their potential contribution to stem cell myogenic commitment. MethodsA commercial blood filter was physically and chemically studied to understand its morphological characteristics and composition. PBMCs were concentrated using this system, further isolated by Ficoll-Paque density gradient centrifugation, and then added in an upper transwell chamber to a 2D co-culture of hBM-MSCs and hSkMs. Myogenic commitment was investigated by RT-PCR, immunofluorescence, and flow cytometry immunophenotyping. Cytokine levels were monitored by ELISA assay in culture media. ResultsThe blood filtration system was disassembled and appeared to be formed by twelve membranes of poly-butylene terephthalate fibers (diameters, 0.9–4.0 μm) with pore size distribution of 1–20 μm. Filter functional characterization was achieved by characterizing collected cells by flow cytometry. Subsequently, collected PBMCs fraction was added to an in-vitro model of hBM-MSC myogenic commitment. In the presence of PBMCs, stem cells significantly upregulated myogenic genes, such as Desmin and MYH2, as confirmed by qRT-PCR and expressed related proteins by immunofluorescence (IF) assay, while downregulated pro-inflammatory cytokines (IL12A at day 14) along the 21 days of culture. NoveltyOur work highlights chemical-physical properties of commercial blood filter and suggests that blood filtrated fraction of PBMC might modulate cytokine expression in response to muscle injury and promote myogenic events, supporting their clinical use in autologous transplantation.

STAT6 suppression prevents bleomycin-induced dermal fibrosis.

Fibrosis of the skin and internal organs is a hallmark of systemic sclerosis (SSc). Although the pathogenesis of SSc is poorly understood, increasing evidence suggests that interleukins (IL)-4 and - 13 contribute to the pathogenesis of skin fibrosis by promoting collagen production and myofibroblast differentiation. Signal transducers and activators of transcription 6 (STAT6) is one of the most important downstream transcription factors activated by both IL-4 and IL-13. However, it is not completely understood whether STAT6 plays a role during the pathogenesis of skin fibrosis in SSc. In this study, we observed increased STAT6 phosphorylation in fibrotic skin samples collected from SSc patients as well as bleomycin-injected murine mice. Knockout of Stat6 in mice significantly (1) suppressed the expression of fibrotic cytokines including Il13, Il17, Il22, Ccl2, and the alternatively activated macrophage marker Cd206; (2) reduced the production of collagen and fibronectin, and (3) attenuated late-stage skin fibrosis and inflammation induced by bleomycin. Consistently, mice treated with STAT6 inhibitor AS1517499 also attenuated skin fibrosis on day 28. In addition, a co-culture experiment demonstrated that skin epithelial cells with STAT6 knockdown had reduced cytokine expression in response to IL-4/IL-13, and subsequently attenuated fibrotic protein expression in skin fibroblasts. On the other side, STAT6 depletion in skin fibroblasts attenuated IL-4/IL-13-induced cytokine and fibrotic marker expression, and reduced CXCL2 expression in co-cultured keratinocytes. In summary, our study highlighted an important yet not fully understood role of STAT6 in skin fibrosis by driving innate inflammation and differentiation of alternatively activated macrophages in response to injury.

MAPLE Processed Nanostructures for Antimicrobial Coatings

Despite their great benefits for debilitated patients, indwelling devices are prone to become easily colonized by resident and opportunistic microorganisms, which have the ability to attach to their surfaces and form highly specialized communities called biofilms. These are extremely resistant to host defense mechanisms and antibiotics, leading to treatment failure and device replacement, but also to life-threatening complications. In this study, we aimed to optimize a silica (SiO2)-coated magnetite (Fe3O4)-based nanosystem containing the natural antimicrobial agent, eugenol (E), suitable for MAPLE (matrix-assisted pulsed laser evaporation) deposition as a bioactive coating for biomedical applications. X-ray diffraction, thermogravimetric analysis, Fourier-transform infrared spectroscopy, and transmission electron microscopy investigations were employed to characterize the obtained nanosystems. The in vitro tests evidenced the superior biocompatibility of such nanostructured coatings, as revealed by their non-cytotoxic activity and ability to promote cellular proliferation and sustain normal cellular development of dermal fibroblasts. Moreover, the obtained nanocoatings did not induce proinflammatory events in human blood samples. Our studies demonstrated that Fe3O4 NPs can improve the antimicrobial activity of E, while the use of a SiO2 matrix may increase its efficiency over prolonged periods of time. The Fe3O4@SiO2 nanosystems showed excellent biocompatibility, sustaining human dermal fibroblasts' viability, proliferation, and typical architecture. More, the novel coatings lack proinflammatory potential as revealed by the absence of proinflammatory cytokine expression in response to human blood sample interactions.

Stress response protein REDD1 promotes diabetes-induced retinal inflammation by sustaining canonical NF-κB signaling

Inflammation contributes to the progression of retinal pathology caused by diabetes. Here, we investigated a role for the stress response protein regulated in development and DNA damage response 1 (REDD1) in the development of retinal inflammation. Increased REDD1 expression was observed in the retina of mice after 16-weeks of streptozotocin (STZ)-induced diabetes, and REDD1 was essential for diabetes-induced pro-inflammatory cytokine expression. In human retinal MIO-M1 Müller cell cultures, REDD1 deletion prevented increased pro-inflammatory cytokine expression in response to hyperglycemic conditions. REDD1 deletion promoted nuclear factor erythroid-2-related factor 2 (Nrf2) hyperactivation; however, Nrf2 was not required for reduced inflammatory cytokine expression in REDD1-deficient cells. Rather, REDD1 enhanced inflammatory cytokine expression by promoting activation of nuclear transcription factor κB (NF-κB). In WT cells exposed to tumor necrosis factor α (TNFα), inflammatory cytokine expression was increased in coordination with activating transcription factor 4 (ATF4)-dependent REDD1 expression and sustained activation of NF-κB. In both Müller cell cultures exposed to TNFα and in the retina of STZ-diabetic mice, REDD1 deletion promoted inhibitor of κB (IκB) expression and reduced NF-κB DNA-binding activity. We found that REDD1 acted upstream of IκB by enhancing both K63-ubiquitination and auto-phosphorylation of IκB kinase complex. In contrast with STZ-diabetic REDD1+/+ mice, IκB kinase complex autophosphorylation and macrophage infiltration were not observed in the retina of STZ-diabetic REDD1-/- mice. The findings provide new insight into how diabetes promotes retinal inflammation and support a model wherein REDD1 sustains activation of canonical NF-κB signaling.

Reprograming of Gene Expression of Key Inflammatory Signaling Pathways in Human Peripheral Blood Mononuclear Cells by Soybean Lectin and Resveratrol.

Inflammation is linked to several human diseases like microbial infections, cancer, heart disease, asthma, diabetes, and neurological disorders. We have shown that the prototype inflammatory agonist LPS modulates the activity of Ubiquitin-Proteasome System (UPS) and regulates transcription factors such as NF-κB, leading to inflammation, tolerance, hypoxia, autophagy, and apoptosis of cells. We hypothesized that proteasome modulators resveratrol and soybean lectin would alter the gene expression of mediators involved in inflammation-induced signaling pathways, when administered ex vivo to human peripheral blood mononuclear blood cells (PBMCs) obtained from normal healthy controls. To test this hypothesis, analysis of RNA derived from LPS-treated human PBMCs, with or without resveratrol and soybean lectin, was carried out using Next Generation Sequencing (NGS). Collectively, the findings described herein suggest that proteasome modulators, resveratrol (proteasome inhibitor) and lectins (proteasome activator), have a profound capacity to modulate cytokine expression in response to proteasome modulators, as well as expression of mediators in multiple signaling pathways in PBMCs of control subjects. We show for the first-time that resveratrol downregulates expression of mediators involved in several key signaling pathways IFN-γ, IL-4, PSMB8 (LMP7), and a subset of LPS-induced genes, while lectins induced IFN-γ, IL-4, PSMB8, and many of the same genes as LPS that are important for innate and adaptive immunity. These findings suggest that inflammation may be influenced by common dietary components and this knowledge may be used to prevent or reverse inflammation-based diseases.

Inflammation triggers ILC3 patrolling of the intestinal barrier

An orchestrated cellular network, including adaptive lymphocytes and group 3 innate lymphoid cells (ILC3s), maintains intestinal barrier integrity and homeostasis. T cells can monitor environmental insults through constitutive circulation, scanning tissues and forming immunological contacts, a process named immunosurveillance. In contrast, the dynamics of intestinal ILC3s are unknown. Using intravital imaging, we observed that villus ILC3s were largely immotile at steady state but acquired migratory ‘patrolling’ attributes and enhanced cytokine expression in response to inflammation. We showed that T cells, the chemokine CCL25 and bacterial ligands regulated intestinal ILC3 behavior and that loss of patrolling behavior by interleukin-22 (IL-22)-producing ILC3s altered the intestinal barrier through increased epithelial cell death. Collectively, we identified notable differences between the behavior of ILC3s and T cells, with a prominent adaptation of intestinal ILC3s toward mucosal immunosurveillance after inflammation.

STING protects breast cancer cells from intrinsic and genotoxic-induced DNA instability via a non-canonical, cell-autonomous pathway.

STING (Stimulator of Interferon Genes) is an endoplasmic reticulum-anchored adaptor of the innate immunity best known to trigger pro-inflammatory cytokine expression in response to pathogen infection. In cancer, this canonical pathway can be activated by intrinsic or drug-induced genomic instability, potentiating antitumor immune responses. Here we report that STING downregulation decreases cell survival and increases sensitivity to genotoxic treatment in a panel of breast cancer cell lines in a cell-autonomous manner. STING silencing impaired DNA Damage Response (53BP1) foci formation and increased DNA break accumulation. These newly identified properties were found to be independent of STING partner cGAS and of its canonical pro-inflammatory pathway. STING was shown to partially localize at the inner nuclear membrane in a variety of breast cancer cell models and clinical tumor samples. Interactomics analysis of nuclear STING identified several proteins of the DNA Damage Response, including the three proteins of the DNA-PK complex, further supporting a role of STING in the regulation of genomic stability. In breast and ovarian cancer patients that received adjuvant chemotherapy, high STING expression is associated with increased risk of relapse. In summary, this study highlights an alternative, non-canonical tumor-promoting role of STING that opposes its well-documented function in tumor immunosurveillance.

Anti-inflammatory and neuroprotective properties of the corticosteroid fludrocortisone in retinal degeneration

The pathogenesis of outer retinal degenerations has been linked to the elevation of cytokines that orchestrate pro-inflammatory responses within the retinal milieu, and which are thought to play a role in diseases such as geographic atrophy (GA), an advanced form of AMD. Here we sought investigate the anti-inflammatory and mechanistic properties of fludrocortisone (FA), as well as triamcinolone acetonide (TA), on Müller cell-mediated cytokine expression in response to inflammatory challenge. In addition, we investigated the neuroprotective efficacy of FA and TA in a photo-oxidative damage (PD), a model of outer retinal degeneration. Expression of CCL2, IL-6, and IL-8 with respect to FA and TA were assessed in Müller cells in vitro, following simulation with IL-1β or TNF-α. The dependency of this effect on mineralocorticoid and glucocorticoid signaling was also interrogated for both TA and TA via co-incubation with steroid receptor antagonists. For the PD model, C57BL/6 mice were intravitreally injected with FA or TA, and changes in retinal pathology were assessed via electroretinogram (ERG) and optical coherence tomography (OCT). FA and TA were found to dramatically reduce the expression of CCL2, IL-6, and IL-8 in Müller glia in vitro after inflammatory challenge with IL-1β or TNF-α (P < 0.05). Though FA acts as both a mineralocorticoid and glucocorticoid receptor agonist, co-incubation with selective steroid antagonists revealed that the suppressive effect of FA on CCL2, IL-6, and IL-8 expression is mediated by glucocorticoid signaling (P < 0.05). In PD, intravitreal FA was found to ameliorate outer-retinal atrophy as measured by ERG and OCT (P < 0.05), while TA had no significant effect (P > 0.05). Our data indicate potent anti-inflammatory and mechanistic properties of corticosteroids, specifically FA, in suppressing inflammation and neurodegeneration degeneration associated with outer retinal atrophy. Taken together, our findings indicate that corticosteroids such as FA may have value as a potential therapeutic for outer retinal degenerations where such pro-inflammatory factors are implicated, including AMD.

Age-dependent effects of the recombinant spike protein/SARS-CoV-2 on the M–CSF– and IL-34-differentiated macrophages in vitro

The SARS-CoV-2 virus causes elevated production of senescence-associated secretory phenotype (SASP) markers by macrophages. SARS-CoV-2 enters macrophages through its Spike-protein aided by cathepsin (Cat) B and L, which also mediate SASP production. Since M-CSF and IL-34 control macrophage differentiation, we investigated the age-dependent effects of the Spike-protein on SASP-related pro-inflammatory-cytokines and nuclear-senescence-regulatory-factors, and CatB, L and K, in mouse M–CSF– and IL-34-differentiated macrophages. The Spike-protein upregulated SASP expression in young and aged male M–CSF–macrophages. In contrast, only young and aged male IL-34-macrophages demonstrated significantly reduced pro-inflammatory cytokine expression in response to the Spike-protein in vitro. Furthermore, the S-protein elevated CatB expression in young male M–CSF–macrophages and young female IL-34-macrophages, whereas CatL was overexpressed in young male IL-34- and old male M–CSF–macrophages. Surprisingly, the S-protein increased CatK activity in young and aged male M–CSF–macrophages, indicating that CatK may be also involved in the COVID-19 pathology. Altogether, we demonstrated the age- and sex-dependent effects of the Spike-protein on M-CSF and IL-34-macrophages using a novel in vitro mouse model of SARS-CoV-2/COVID-19.

Chloroquine potentially modulated innate immune response to Vibrio parahaemolyticus in RAW 264.7 macrophages

ABSTRACTVibrio parahaemolyticus, as a vibriosis, causes huge losses to the aquaculture industry, food poisoning in humans and activates macrophages to induce pro-inflammatory cytokines. Chloroquine (CQ), as an anti-inflammatory property, this study aimed to investigate the effect of CQ on inflammatory response to V. parahaemolyticus in RAW 264.7 macrophages. The result showed that V. parahaemolyticus induced the production of T-cell co-stimulatory molecules (CD80, CD86, CD40 and MHC II), and increased the production and mRNA expression of pro-inflammatory, anti-inflammatory cytokines and type I interferons (IFNs). Meanwhile, the stimulated macrophages elevated cellular stress-released reactive oxygen species and nitric oxide synthase to trigger an antimicrobial response, and effectively reduced the accumulation of acetylated low-density lipoprotein and neutral lipids. Finally, we found that chloroquine (CQ) was involved in inhibiting inflammatory cytokine expression in response to V. parahaemolyticus. These results suggested that CQ potentially attenuated the inflammatory cytokine expression in Gram-negative bacterium-activated macrophages.

Neonatal Deficiencies in NK cell-Activating Cytokines in the Context of Bordetella pertussis Infection

Abstract Whooping cough, caused by Bordetella pertussis, has seen a resurgence in recent years, in part due to the switch from the whole cell to acellular vaccine. Although both adults and infants can suffer from severe paroxysmal cough, infants can experience a fatal health decline. Disease progression in infant and adult mouse models show some parallels to human disease. Adult mice exhibit severe lung pathology, but overcome infection, while infant mice experience mild lung pathology, develop leukocytosis, bacterial dissemination, and succumb to infection. We hypothesize that this discrepancy in response to infection is due to a deficiency in neonatal Interferon gamma (IFN-y) production by Natural Killer (NK) cells, which can activate protective T-helper 1 (Th1) cells. We further hypothesize that this deficiency is in part due to the inability of neonatal antigen presenting cells to produce the NK cell-activating cytokines Interleukin (IL) 12 and IL18 when exposed to B. pertussis. To test this hypothesis, we isolated splenocytes from infected and uninfected infant and adult mice, incubated with heat killed B. pertussis, and analyzed for mRNA expression of cytokines via qRT-PCR. While adult splenocytes responded robustly with upregulated expression of IL12 and TNF-α, infant splenocytes failed to upregulate expression of these cytokines in response to bacterial stimulation. Moreover, analysis of lung tissue from infected mice revealed robust upregulation of IL12, IL18 and TNF-α in adult mice but not in infant mice. These results indicate that infant mouse cells fail to upregulate expression of NK cell-activating cytokines in response to B. pertussis infection, leading to a lack of protective IFN-y responses.

Inhibition of activator protein 1 attenuates neuroinflammation and brain injury after experimental intracerebral hemorrhage.

AimsIntracerebral hemorrhage (ICH) is a devastating type of stroke without specific treatment. Activator protein 1 (AP‐1), as a gene regulator, initiates cytokine expression in response to environmental stimuli. In this study, we investigated the relationship between AP‐1 and neuroinflammation‐associated brain injury triggered by ICH.MethodsIntracerebral hemorrhage mice were developed by autologous blood or collagenase infusion. We measured the dynamics of AP‐1 in mouse brain tissues during neuroinflammation formation after ICH. The effects of the AP‐1 inhibitor SR11302 on brain injury and neuroinflammation as well as the underlying mechanisms were investigated in vivo and in vitro.ResultsAP‐1 was significantly upregulated in mouse brain tissue as early as 6 hours after ICH, accompanied by elevations in proinflammatory factors, including interleukin (IL)‐6, IL‐1β, and tumor necrosis factor (TNF)‐α. Inhibition of AP‐1 using SR11302 reduced neurodeficits and brain edema at day 3 after ICH. SR11302 ablated microglial IL‐6 and TNF‐α production and brain‐infiltrating leukocytes in ICH mice. In addition, SR11302 treatment diminished thrombin‐induced production of IL‐6 and TNF‐α in cultured microglia.ConclusionsInhibition of AP‐1 curbs neuroinflammation and reduces brain injury following ICH.

MiR-146a is a negative regulator of IL-33-stimulated mouse mast cells

Abstract Interleukin-33 (IL-33) is an inflammatory cytokine that promotes allergic disease by activating ILC2, Th2, and mast cells. IL-33 is increased in asthmatics, and its blockade suppresses asthma-like inflammation in mouse models. The IL-33 receptor, ST2, shares signaling cascades with the TLR family, but homeostatic control of ST2 function is poorly understood. MicroRNA-146a (miR-146a) is induced by LPS and suppresses TLR4 signaling in macrophages. Therefore, we explored whether miR-146a has a role in IL-33 signaling. IL-33 induced cellular and exosomal miR-146a expression in mouse bone marrow derived mast cells (BMMC). Induction required MyD88, Akt, and NFkB, since antagonizing these pathways decreased miR-146 expression. BMMC transfected with a miR-146a antagomir or derived from miR-146a KO mice showed enhanced cytokine expression in response to IL-33, suggesting that miR-146a is a negative regulator of IL-33-ST2 signaling. Our data further suggest that miR-146a may act by targeting IRAK proteins, because mir-146a KO BMMC have increased TRAF6, IRAK1, and IRAK4 expression. In vivo, miR-146a expression in plasma exosomes was elevated after intraperitoneal injection of IL-33. Also mast cell deficient c-Kitw-sh mice acutely reconstituted with miR-146a KO BMMC intraperitoneally had elevated plasma IL-6 levels in comparison to their WT counterparts after IL-33 challenge. These results support the hypothesis that miR-146a is a feedback regulator of IL-33-mediated mast cell functions associated with allergic disease.

The Single Nucleotide Polymorphism Mal-D96N Mice Provide New Insights into Functionality of Mal in TLR Immune Responses.

MyD88 adaptor-like (Mal) protein is the most polymorphic of the four key adaptor proteins involved in TLR signaling. TLRs play a critical role in the recognition and immune response to pathogens through activation of the prototypic inflammatory transcription factor NF-κB. The study of single nucleotide polymorphisms in TLRs, adaptors, and signaling mediators has provided key insights into the function of the corresponding genes but also into the susceptibility to infectious diseases in humans. In this study, we have analyzed the immune response of mice carrying the human Mal-D96N genetic variation that has previously been proposed to confer protection against septic shock. We have found that Mal-D96N macrophages display reduced cytokine expression in response to TLR4 and TLR2 ligand challenge. Mal-D96N macrophages also display reduced MAPK activation, NF-κB transactivation, and delayed NF-κB nuclear translocation, presumably via delayed kinetics of Mal interaction with MyD88 following LPS stimulation. Importantly, Mal-D96N genetic variation confers a physiological protective phenotype to in vivo models of LPS-, Escherichia coli-, and influenza A virus-induced hyperinflammatory disease in a gene dosage-dependent manner. Together, these results highlight the critical role Mal plays in regulating optimal TLR-induced inflammatory signaling pathways and suggest the potential therapeutic advantages of targeting the Mal D96 signaling nexus.