ObjectivesCRISPR/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.MethodsThe 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.ResultsWe 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.ConclusionThe 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.

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.

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.

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.

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.

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.

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.

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