OBJECTIVES:CRISPR/Cas9 is currently the primary tool used for genome editing in mammalian cells. To cleave and alter genomic DNA, both the Cas9 nuclease and a guide RNA (gRNA) must be present in the nucleus. One preferred method of introducing these reagents is direct transfection of a recombinant Cas9 protein complexed with a synthetic gRNA as a ribonucleoprotein (RNP) complex. It is well established from prior work in RNA interference that synthetic RNAs can induce a type I interferon (IFN) response that can limit the application of such methods both in vitro and in vivo. While the immunological properties of short siRNAs are well understood, little is known about the immune recognition of longer CRISPR gRNAs. The objective of our in vitro study was to investigate how the composition of the gRNA influences its recognition by human immune cells.METHODS:The study was performed in vitro in human peripheral blood mononuclear cells (PBMCs). The PBMCs from healthy donor volunteers were treated with gRNA for 24 h, and the levels of type I IFNs in culture supernatants were measured by a multiplex enzyme-linked immunosorbent chemiluminescent assay. Prior to the analysis in PBMCs, the physicochemical parameters and functionality of all nucleic acid constructs were confirmed by electrospray-ionization mass spectrometry and CRISPR/Cas9 gene editing assessment in HEK293-Cas9 cells, respectively.RESULTS:We found that unmodified synthetic CRISPR gRNAs triggered a strong IFN response in PBMC cultures in vitro that could be prevented with chemical modification. Likewise, in vitro-transcribed single-guide RNAs (sgRNAs) also triggered a strong IFN response that could only be partially suppressed by phosphatase removal of the 5'-triphosphate group. However, the process by which the gRNA is prepared (i.e., chemically synthesized as a two-part crRNA:tracrRNA complex or in vitro-transcribed as an sgRNA) does not directly influence the immune response to an unmodified gRNA. When experiments were performed in the HEK293 cells, only in vitro-transcribed sgRNA containing 5'-triphosphate induced IFN secretion.CONCLUSION:The results of our structure-activity relationship study, therefore, suggest that chemical modifications commonly used to reduce the immunostimulation of traditional RNA therapeutics can also be used as effective tools to eliminate undesirable IFN responses to gRNAs.
Objective: We have previously shown changes in protein, immune cell, nitric oxide a neurotrophic factor content and distribution in the olfactory bulb of an endotoxin-treated rat model, as all these factors have been shown to be involved in neurodegeneration. We expanded our studies by fluorescently imaging smooth muscle actin and endothelial nitric oxide synthase (eNOS), both markers of blood vessels, to investigate loss of vasculature content, as well as imaging locations and quantities of immune cells. The work was performed to shed further light on associations between vessel integrity and immune cell initiated endothelial disruption. Our goal was to demonstrate that cytokine production, NOS induction and immune cell increases, are likely part of the process that leads to a loss of olfaction and dopaminergic signaling and includes vascular perturbations. Methods: Rats were sacrificed following lipopolysaccharide (LPS) treatment. Olfactory bulbs were harvested, sectioned from top to bottom to include the tract and sensory neurons, and probed for markers of inflammation. Inducible nitric oxide synthase (iNOS), neuronal nitric oxide synthase (nNOS), eNOS, interleukin-1 beta (IL-1β), TNF-α, interleukin-6, glial cell derived neurotrophic factor (GDNF) and circulating nitric oxide (NO) were imaged together with tagged macrophages, T-cells, B-cells and neutrophils. Results: Serum NO levels indicated that an inflammatory episode had occurred, being significantly higher in treated animals, with tissue levels of NOS elevated for an extended period of time. Immune cell clusters were seen in a number of areas and the localization of NOS isomers suggests that they have divergent roles in neurodegeneration. For instance, eNOS was associated with blood vessels, iNOS with glial and matrix cells and nNOS with glial cells and neurons. T and B-cell numbers showed a sustained increase; neutrophil numbers rapidly increased then returned to baseline levels; macrophage numbers increased and remained high; LAMP positive cell numbers (NK-cells) increased and remained high; GDNF content increased; IL-6, TNF-α and IL-1β levels all rapidly increased, before dropping to untreated levels, while circulating, NO levels increased dramatically. Of interest, the images of vascular content, immune cell content, eNOS and smooth muscle actin, allowed us to show detrimental interactions between cells, factors and vessels. Our data show that the majority of the vessels were intact, though sections of interest were ‘extracted’ to reveal possible leaky areas. Specific sites of IL-6 positive lymphocyte clustering were noted around vessels, suggesting that interactions are occurring that lead to disruptions of blood vessel tunicae, allowing the internalization of circulating cells and subsequent cytokine-initiated endothelial cell death. Conclusion: Our findings suggest that protective GDNF and eNOS, which maintains vascular tone, are possibly synthesized too late to combat cytokine initiated neuron damage, glial activation and chronic loss of vascular integrity.
Triptolide, a Chinese plant medicine from Tripterygium wilfordii Hook F, has been shown to have inhibitory effects on macrophage activities, and we recently found that it induces sudden macrophage cell death in the presence of bacterial lipopolysaccharide (LPS). In this present study, we examined precise mechanisms underlying induction of the cytotoxicity of triptolide toward macrophages in relation to the action of LPS, and thereby showed that the cytotoxic effects depended on the concentrations of both triptolide and LPS. More than 10 ng/mL LPS was necessary in combination with 300 ng/mL triptolide. However, pre-treatment with 1 ng/mL LPS for 60 min abolished the cytotoxicity induced by 100 ng/mL LPS and 300 ng/mL triptolide, showing that the cytotoxicity was regulated by LPS-tolerance. Besides, in primary macrophages obtained from mouse peritoneum, those from C3H/HeN mice, an LPS-responder, showed similar susceptibility to triptolide and LPS-induced cell damage; whereas those from C3H/HeJ mice, an LPS hypo-responder, did not, suggesting that the cytotoxic effect of triptolide was linked to the LPS/Toll-like receptor 4 (TLR4)-signaling cascade. These results suggest that the cytotoxicity of triptolide toward macrophages was regulated by the LPS-signaling cascade through both down-regulation known as LPS-tolerance and the TLR4 receptor.
The first objective of this study was to identify if soluble tumor necrosis factor-receptor 1 (sTNFr1) and -receptor 2 (sTNFr2) are modulated by vitamin D status (insufficient vs. sufficient). The second objective was to reveal if soluble TNF receptors fluctuate with serum 25-hydroxyvitamin D (25(OH)D) concentrations following a bolus of supplemental vitamin D. Reportedly healthy male adults were randomly (double-blind) assigned to a placebo (n=15) or vitamin D (100,000 IU of cholecalciferol; n=14) supplement. Supplements were taken as a bolus immediately after and on the same day as providing the first blood sample (baseline (Bsl)). Fasting blood samples were also obtained at 1-, 3-, 7-, and 24-d after the bolus. Serum 25(OH)D, 1,25-dihyroxyvitamin D (1,25(OH)D), tumor necrosis factor (TNF)-α, sTNFr1, and sTNFr2 were measured in each blood sample. At Bsl, subjects were classified as vitamin D insufficient (serum 25(OH)D
The nuclear receptor aryl hydrocarbon receptor (AhR) plays a crucial role in receptor activator of nuclear factor (NF)-kB ligand (RANKL)-mediated osteoclastogenesis. A considerable amount of evidence reveals that AhR is also a key modulator of skeletal remodeling. However, the mechanism underlying the regulation of AhR expression in osteoclasts and the signaling pathway through which AhR controls osteoclastogenesis remain unclear. We found that the expression of AhR in bone marrow-derived osteoclasts was upregulated by RANKL at an earlier stage than the expression of signature osteoclast genes such as NFATc1, c-Src, integrin avb3, and cathepsin K. In response to RANKL, bone marrow macrophages isolated from AhR−/− mice exhibited impaired phosphorylation of Akt and mitogen-activated protein kinase as well as NF-kB, while their response to the macrophage colony-stimulating factor remained unchanged. Osteoclast differentiation mediated by the AhR signaling pathway was also regulated in a RANKL/c-Fos-dependent manner. Further, ligand activation of AhR by the smoke toxin benzo [a] pyrene (BaP) accelerated osteoclast differentiation in a receptor-dependent manner, and AhR-dependent regulation of mitochondrial biogenesis in osteoclasts was observed. Moreover, AhR−/− mice exhibited impaired bone healing with delayed endochondral ossification. Taken together, the present results suggest the new role that the RANKL-AhR-c-Fos signaling axis plays a critical role in osteoclastogenesis, thereby identifying the potential of AhR in treating pathological, inflammatory, or metabolic disorders of the bone.
Objective: Macrophages play a role in clearing bacteria from the gut in the initial stages of necrotizing enterocolitis (NEC). Macrophage Inflammatory Protein-1α (MIP-1α), also known as CCL3, is a chemokine produced by macrophages that enhances the immune response. It acts as both a recruiter of immune cells and a coactivator of other macrophages. Interleukin-12 (IL-12) is a pro-inflammatory chemotactant produced by several types of phagocytic cells such as dendritic cells and neutrophils, but primarily by macrophages. We hypothesized that the pro-inflammatory state associated with NEC pathophysiology would induce increased expression of MIP-1α and IL-12 that would be detectable in serum and intestinal tissue. Methods: Timed pregnant Sprague-Dawley rats were randomized by litter. Controls were delivered vaginally and dam-fed. NEC pup groups were delivered 12 h prematurely via Cesarean section, formula fed, given a single oral dose of lipopolysaccharide, and subjected to intermittent cold and hypoxia as part of a proven NEC rat model protocol. Animals were sacrificed at 0, 12, 24, 48, 72, and 96 h of life and serum and intestinal tissue samples were collected. Samples were analysed via western blot using antibodies with affinity to MIP-1α and IL-12 respectively. Results: Serum and ileal levels of MIP-1 alpha were increased from 48 to 72 h in NEC animals when compared with controls. Serum and ileal IL-12 was downregulated in NEC groups at 12 and 24 h compared to controls. Conclusion: Macrophage function plays an important role in the first 48 h of NEC pathophysiology. Downregulation of IL-12 in the setting of increased MIP-1α expression may represent deranged or inhibited macrophage function in the context of NEC pathogenesis. Further work to elucidate the significance of these findings is warranted.
Objective: Anti-TNF-α therapy is an effective therapy for Crohn’s disease (CD). Thus anti-TNF-α therapy may alter CD64 expression, one of the Fc receptors for IgG, in CD. We investigated CD64 expression on neutrophils before and after treatment with anti-TNF-α therapy in CD. Methods: A total of 11 patients with active CD treated with anti-TNFα antibodies were enrolled. The severity of CD was assessed with the CD activity index (CDAI). Peripheral venous blood was obtained before and 2 weeks after the initial administration of anti-TNF-α antibody. CD64 expression on neutrophils was measured by FACS analysis of whole blood samples. Results: CDAI, C-reactive protein value and CD64 expression decreased significantly after anti-TNF-α therapy. Both prior to and after anti-TNF treatment, there was a significant and positive correlation between CD64 expression and CDAI or CRP. Similarly, there was a significant and negative correlation between CD64 and albumin value. Conclusion: Anti-TNF-α therapy suppresses CD64 expression of neutrophils, which may account for the mechanism underlying the efficacy of the medication in CD.
The recent discovery of group 2 innate lymphoid cells (ILC2s) has caused a paradigm shift in the understanding of allergic airway disease pathogenesis. Prior to the discovery of ILC2s, Th2 cells were largely thought to be the primary source of type 2 cytokines; however, activated ILC2s have since been shown to contribute significantly, and in some cases, dominantly to type 2 cytokine production. Since the discovery of ILC2s in 2010, many mediators have been shown to regulate their effector functions. Initial studies identified the epithelial derived cytokines IL-25, IL-33, and TSLP as activators of ILC2s, and recent studies have identified many additional cytokine and lipid mediators that are involved in ILC2 regulation. ILC2s and their mediators represent novel therapeutic targets for allergic airway diseases and intensive investigation is underway to better understand ILC2 biology and upstream and downstream pathways that lead to ILC2-driven airway pathology. In this review, we will focus on the cytokine and lipid mediators that regulate ILC2s in human allergic airway disease, as well as highlight newly discovered mediators of mouse ILC2s that may eventually translate to humans.
In this study, we sought to improve ligament healing by modulating the inflammatory response after acute injury through the neutralization of Interleukin-17 (IL-17), which we hypothesized would decrease inflammatory cell infiltration and cytokine production. Administration of an Interleukin-17 neutralizing antibody (IL-17 NA) immediately following a rat medial collateral ligament (MCL) transection resulted in alterations in inflammatory cell populations and cytokine expression within the healing ligament, but did not reduce inflammation. Specifically, treatment resulted in a decrease in M2 (anti-inflammatory) macrophages, an increase in T cells, and an increase in the levels of IL-2, IL-6, and IL-12 in the MCL 7 days post injury. IL-17NA treatment, and subsequent immunomodulation, did not result in improved ligament healing, as measured by collagen composition and wound size.
A normal healing response after ligament and tendon rupture results in scar formation and an inferior tissue that fails to emulate its original structure, composition, and function. More regenerative healing (closer to the original) can be obtained through early suppression of inflammatory cells and associated cytokines. Examination of the immune mediated response of mesenchymal stem/stromal cells (MSCs) during healing indicates that MSCs reprogram macrophages from a pro-inflammatory M1 phenotype to an anti-inflammatory M2 phenotype. Based on these studies our objective was to treat ligament and tendon injuries with MSCs in order to modulate their inflammatory response. Our initial studies using allogeneic cells demonstrated an in vivo dose dependency of MSCs on ligament healing. Medial collateral ligaments (MCLs) treated with 1 × 106 (low dose) MSCs exhibited less inflammation and a reduced number of M1 macrophages compared to ligaments treated with 4 × 106 (high dose) MSCs. Strength of ligament was also improved with the low dose treatment. We then examined the in vivo effects of MSCs that had been preconditioned to be more anti-inflammatory. Treatment with these preconditioned MSCs was compared with normally processed (unconditioned) MSCs using the rat Achilles tendon and MCL healing models. Pre-conditioned MSCs significantly reduced inflammation by increasing the M2 macrophages and decreasing the M1 macrophages. Most importantly, treatment with pre-conditioned MSCs improved tissue strength to levels comparable to intact tissue. Overall, pre-conditioned MSC-treatment out-performed unconditioned MSCs to improve ligament and tendon healing by stimulating a more robust, paracrine-mediated immunosuppressive response.
Polysaccharides or heteroglycan isolated from fungal species have attained wide recognition for their immune modulating functions. However, there exists a significant relationship in the structural and functional activity of this molecule. In this commentary, we focus on a well characterized heteroglycan isolated from the mycelia of Pleurotus ostreatus which have been shown to drive immune stimulation in tumor bearing mice. The heteroglycan possessed high molecular weight and was primarily composed of glucose, mannose and fucose in a 3:2:1 ratio with major β- linkages in the repeating chain. The molecule also exhibited triple helical conformation in solutions. These immune driving molecules have a significant impact in decreasing the tumor volume in model mice and they are known to play a significant role in the proliferation and activation of lymphocytes in the tumor bearing mice. The physical traits of the heteroglycan were associated with its immune stimulating ability and such compounds can therefore be utilized as adjuvants in immune based therapies.
Only primates suffer from hyperuricemia and gout. Acute gouty attack with fulminate inflammation may recover within 7-10 days. The pathogenesis of monosodium urate monohydrate (MSU) crystal-induced acute gouty inflammation has been gradually elucidated. It is conceivable that MSU crystals possess both danger-associated pathological pattern (DAMP) and physical microcrystal properties that may activate innate immune cells IL-1 production and subsequent IL-6, IL-8 and TNF-α release from neighborhood cells. By contrast, the molecular basis of acute gouty inflammation resolution mostly remains unclear. Previous studiesdemonstrated that intracellular negative cytokine regulators CIS and SOCS-3 involved in the resolution of acute gouty inflammation in synergism with anti-inflammatory cytokine molecules (TGF-β1, IL-10 and soluble TNF-α receptors type 1 and 2). This commentary aims to dissect the potential mechanisms of induction and resolution of acute gouty attack. Besides, the unsolved problems in these issues are discussed.
Macrophages play an important role in immune and inflammatory responses, and have been extensively studied in vitro using culture media such as RPMI1640 medium, Dulbecco's modified Eagle medium (DMEM), and Ham's F-12 medium (F-12). We found that the activation phenotypes of a murine macrophage-like cell line, J774.1/JA-4, were obviously different in two distinct culture media (F-12 and DMEM), both of which were supplemented with 10% of the same fetal bovine serum (FBS). Among these phenotypes, nitric oxide (NO) production as well as inducible NO synthase (iNOS) expression, induced by lipopolysaccharide (LPS) and interferon-γ (IFN-γ), were remarkably different. iNOS expression was higher in the macrophages cultured in DMEM than in F-12 for 20h, while no significant differences were shown in NO production between in F-12 and DMEM. It might be the reason why DMEM have reduced NO production by the induced iNOS. Besides, O2−-generating activity, and production of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in the activated macrophages were also different between the cultures in F-12 and DMEM. These results suggest that F-12 and DMEM contain certain components responsible for modification of macrophage activation processes and/or macrophage functions. Our present results provide evidence that the choice of culture medium is important in the study and analysis of macrophage activation.
Interleukin 2 (IL-2) is a monomeric glycoprotein that is primarily produced by activated CD4+ T cells, CD8+ T cells and dendritic cells. It is characterized as a proinflammatory cytokine that is secreted by Th1 cells. IL-2 plays a central role in the activation of regulatory T cells to produce the cytokines tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ). IL-2 may also enhance the cytolytic activity of natural killer cells, thereby ensuring their significance in the control of the immune response, and effectively participate in the pathogenesis of several pathological conditions, such as cancer and metabolic, infectious, autoimmune and inflammatory diseases. We emphasize the importance of studies of IL-2 and discuss perspectives resulting from our increasing understanding of genetic diversity and its role in the immune response.
Obesity is dramatically increasing worldwide and there exists a significant association of its prevalence with Type 2 Diabetes Mellitus, hypertension, cardiovascular diseases etc. The altered homeostasis of nutritionally overloaded metabolic cells marks the development of obesity induced inflammation. It is marked by elevated expression of the genes encoding for cytokines, chemokines and other inflammatory mediators through activated transcription factorsnuclear factor-kB, activator protein-1, nuclear factor of activated T cells and signal transducer and activator of transcription 3 and; execution of a macromolecular innate immune cell sensor- inflammasome to activate caspase-1 pathway resulting in photolytic maturation. In brief, there occurs an evidential increase in pro-inflammatory cytokines released from the M1 macrophages of white adipose tissue including TNF-α, IL-6, CRP, IL-1β, etc. on contrary an observable decrease in anti-inflammatory cytokines like IL-10, IL-Ra, adiponectin etc. released from M2 macrophages. Along with adipose tissue, immune cells, liver, brain, muscles and pancreas also suffers undergoes inflammatory damage. The inflammatory kinases like JNK and IKK apart from inhibiting insulin action and glucose uptake, also down-regulate transcriptional process resulting in increased expression of pro-inflammatory cytokines. The inflammatory process is initiated by Macrophage-like Kupffer cells following the transducer signals produced by the white adipose tissues further leading to necro-inflammation. Muscle fibre is said to suffer from decreased glycogen synthesis. Obesity also triggers the hypothalamic-pituitary adrenal axis. Pancreas modulating the insulin homeostasis and glucose tolerance is also exaggeratedly affected. Multi-dimensional interventions so as to check obesity induced metaflammation have been undertaken that includes therapeutic intervention and administration of synthetic drugs to target the actual inflammatory. Thus, by proper weight management and diet inflammatory responses in obesity can be controlled to a large extent.
Objective: The objective of the study was to investigate concentrations of cytokines in peritoneal fluid from women with infertility and chronic infectious inflammation (CII) of reproductive system. Methods: 65 patients were included to the study: 50 patients were diagnosed with tubo-peritoneal infertility and CII of reproductive system in remission (CII was of bacterial origin); 15 patients were almost healthy age-matched woman. The concentrations of TNF-α, IL-4, IL-6, IL-10, monocyte chemo attractant protein-1 (MCP-1), IFN-γ and sVCAM-1 were measured in peritoneal fluid using enzyme-linked immunoassay. Results: Peritoneal fluid levels of TNF-α, IL-4, IL-6, IFN-γ, MCP-1, sVCAM-1 were significantly (p<0.05) higher in patients with infertility and CII compared to control group. The most significant difference (4.52 times) was found for IFN-γ concentrations. The concentration of IL-10 was not significantly differing between the groups. Conclusions: Local inflammation and activation of immune reactions with cytokines imbalance in patients with CII of reproductive system may play a role in the pathogenesis of tubo-peritoneal infertility.
Macrophages are heterogeneous cells with diverse phenotypes and sometimes opposing functions. These activities are dictated by activating stimuli in their microenvironment. For example, it is well described how CD4 + T helper (Th) cell-derived cytokines result in different macrophage-activation states. However, much less is known on how differentially-activated macrophages, presenting antigen, can drive the major types of CD4 + Th subpopulations, especially in human systems. Many studies have focussed on dendritic cells as the major antigen-presenting cell shaping T cell responses or on murine macrophage-secreted cytokines in the presence of mitogenic-stimuli, such as CD3/CD28, to induce Th polarization. Recent literature is, however, providing evidence that activated antigen- presenting macrophages can be as efficient as dendritic cells in polarising Th cells, especially Th17, and whilst both these cell types co-exist within inflamed tissue, macrophages are more abundant. The bias towards polarization of particular T cell subsets is strongly dependent on the activation state of macrophages. The concept of targeting macrophages to downregulate inflammatory responses may therefore have further reaching consequences by also abrogating pathogenic Th cells in autoimmune or inflammatory diseases.
Lipoxygenases (LOX) are lipid-peroxidizing enzymes that play a role in cell differentiation, but have also been implicated in the pathogenesis of inflammatory, hyperproliferative and neurological disorders. They are widely distributed in plants and mammals but also occur sporadically in lower organisms. The human genome involves six functional LOX genes and a corrupted pseudogene. 20 years ago it was reported that expression of ALOX15 was specifically induced in human peripheral monocytes by the classical Th2 cytokine interleukin 4 and later expression array profiles indicated that this enzyme is the most strongly upregulated gene product in human monocytes. Although the molecular basis for this IL4-dependent expression regulation has extensively been studied during the past 20 years, there are still a number of unsolved questions. This review is aimed at summarizing the current knowledge on the cytokine-dependent expression regulation of ALOX15 with particular focus on the Th2 cytokines interleukin-4 and interleukin-13 in various cells and tissues and at critically evaluating the potential biological implication of this effect.
IL-15 is an immune-enhancing cytokine belonging to the IL-2 family, which supports survival, proliferation and functional activities of NK, NK-T, T and B cells. Moreover, IL-15 may support the growth and survival of different lymphoid malignancies, suggesting that targeting of the IL-15/IL-15R system or its downstream signaling cascade may result in therapeutic effects, in these tumors. On the other hand, given its immune-enhancing activities IL-15 has been considered a good candidate for cancer immunotherapy. Indeed, IL-15 or IL-15 super agonists have shown anti-tumor activity in several animal tumor models either alone or combined with other immune-enhancing molecules. Therefore, clinical trials of IL-15 or IL-15 super-agonists are ongoing in different cancers. Here we will summarize the biological features of the IL-15/IL-15R system and discuss its duality in tumor biology and the potential applications of IL-15 agonists and antagonists in cancer.
