Specific Silencing of Microglial Gene Expression in the Rat Brain by Nanoparticle-Based Small Interfering RNA Delivery.

Microglia are the major innate immune cells in the brain and are essential for maintaining homeostasis in a neuronal microenvironment. Currently, a genetic tool to modify microglial gene expression in specific brain regions is not available. In this report, we introduce a tailor-designed method that uses lipid and polymer hybridized nanoparticles (LPNPs) for the local delivery of small interfering RNAs (siRNAs), allowing the silencing of specific microglial genes in the hypothalamus. Our physical characterization proved that this LPNP-siRNA was uniform and stable. We demonstrated that, due to their natural phagocytic behavior, microglial cells are the dominant cell type taking up these LPNPs in the hypothalamus of rats. We then tested the silencing efficiency of LPNPs carrying a cluster of differentiation molecule 11b (CD11b) or Toll-like receptor 4 (TLR4) siRNA using different in vivo and in vitro approaches. In cultured microglial cells treated with LPNP-CD11b siRNA or LPNP-TLR4 siRNA, we found a silencing efficiency at protein expression levels of 65 or 77%, respectively. In line with this finding, immunohistochemistry and western blotting results from in vivo experiments showed that LPNP-CD11b siRNA significantly inhibited microglial CD11b protein expression in the hypothalamus. Furthermore, following lipopolysaccharide (LPS) stimulation of cultured microglial cells, gene expression of the TLR4 downstream signaling component myeloid differentiation factor 88 and its associated cytokines was significantly inhibited in LPNP-TLR4 siRNA-treated microglial cells compared with cells treated with LPNP-scrambled siRNA. Finally, after LPNP-TLR4 siRNA injection into the rat hypothalamus, we observed a significant reduction in microglial activation in response to LPS compared with the control rats injected with LPNP-scrambled siRNA. Our results indicate that LPNP-siRNA is a promising tool to manipulate microglial activity locally in the brain and may serve as a prophylactic approach to prevent microglial dysfunction-associated diseases.

Effects of myeloid cell-restricted TNF inhibitors in vitro and in vivo.

Systemic TNF neutralization can be used as a therapy for several autoimmune diseases. To evaluate the effects of cell type-restricted TNF blockade, we previously generated bispecific antibodies that can limit TNF secretion by myeloid cells (myeloid cell-specific TNF inhibitors or MYSTIs). In this study several such variable domain (VH) of a camelid heavy-chain only antibody-based TNF inhibitors were compared in relevant experimental models, both in vitro and in vivo. Pretreatment with MYSTI-2, containing the anti-F4/80 module, can restrict the release of human TNF (hTNF) from LPS-activated bone marrow-derived macrophage (BMDM) cultures of humanized TNF knock-in (mice; hTNFKI) more effectively than MYSTI-3, containing the anti-CD11b module. MYSTI-2 was also superior to MYSTI-3 in providing in vivo protection in acute toxicity model. Finally, MYSTI-2 was at least as effective as Infliximab in preventing collagen antibody-induced arthritis. This study demonstrates that a 33 kDa bispecific mini-antibody that specifically restricts TNF secretion by macrophages is efficient for amelioration of experimental arthritis.

Inhibition of Pro-Inflammatory Functions of Human Neutrophils by Constituents of Melodorum fruticosum Leaves.

In an initial screening, the dichloromethane extract from the leaves of Melodorum fruticosum showed distinct inhibitory effects on the release of interleukin-8 (IL-8) in human neutrophils. Therefore, the aim of the present study was the phytochemical and pharmacological investigation of this extract, to better understand which compounds might be responsible for the anti-inflammatory effect. Phytochemical analysis led to the isolation of 12 known compounds and two new natural products, 5-hydroxy-6-(2-hydroxybenzyl)-4',7-dimethoxyflavanone (13) and 2',4'-dihydroxy-3'-(2-hydroxybenzyl)-4,6'-dimethoxychalcone (14). The influence of the isolated compounds on the production and release of the pro-inflammatory factors IL-8, tumor necrosis factor alpha (TNF-α), reactive oxygen species (ROS), and adhesion molecules (CD62L and CD11b) in human neutrophils was evaluated. Three constituents, melodamide A, 2',4'-dihydroxy-4,6'-dimethoxychalcone, and 2',6'-dihydroxy-4'-methoxychalcone, showed significant inhibition of IL-8 release (IC50 =6.6, 8.6, and 11.6 µm, respectively) and TNF-α production (IC50 =4.5, 13.3, and 6.2 µm, respectively).

Anti-CD11b antibody treatment suppresses the osteoclast generation, inflammatory cell infiltration, and autoantibody production in arthritis-prone FcγRIIB-deficient mice.

BACKGROUND: Previously we established an arthritis-prone FcγRIIB-deficient mouse strain (designated KO1). Anti-mouse CD11b mAb (5C6) has been reported to inhibit the recruitment of peripheral CD11b+ myelomonocytic cells from the blood to the inflammatory site. These cells include neutrophils and monocytes, both of which play important roles in the development of arthritis. Here we treated KO1 mice with 5C6 mAb in order to study its effect on arthritis development. METHODS: To evaluate the disease-preventive effect of 5C6, 4-month-old preclinical KO1 mice were divided into three groups: the first treated with 5C6 for 6 months, the second treated with normal rat IgG for 6 months, as a control, and the third left untreated. Arthritis severity and immunological abnormalities were compared among the groups, along with transcriptional levels of several important arthritis-related factors in ankle joints, spleen, and peripheral blood cells. RESULTS: The 5C6 treatment ameliorated arthritis in KO1 mice, showing decreases in inflammatory cell infiltration and osteoclast formation. Analysis of transcriptional levels in ankle joints revealed that compared with the two control groups, the 5C6-treated group showed downregulated expression of RANK, RANKL, MCP-1, RANTES, TNFα, and IL-6, and at the same time showed significantly up-regulated expression of the decoy receptor for RANKL, i.e. osteoprotegerin. In addition, the disease suppression was associated with the lower serum levels of autoantibodies, and the decreased frequencies of activated B cells and plasma cells. The expression levels of B cell activation/differentiation-related cytokines were suppressed in spleen and peripheral leukocytes of the 5C6-treated mice. Intriguingly, while untreated KO1 mice spontaneously developed marked monocytosis, the 5C6-treated mice showed the significantly down-regulated frequency of monocytes. CONCLUSIONS: The outcome of 5C6 treatment was complex, in which the 5C6-mediated disease-preventive effect is likely due on one hand to the decrease in the recruitment of inflammatory cells and osteoclast precursor monocytes from the periphery into the joints, and on the other hand to the suppression of B cell activation/maturation and of autoantibody production via the suppression of B cell stimulating cytokine production. The lower levels of these cytokines may be the secondary effect of the lower frequency of monocytes, since monocytes/macrophages are the major producers of these cytokines.

Malignant Astrocytic Tumor Progression Potentiated by JAK-mediated Recruitment of Myeloid Cells.

Purpose: While the tumor microenvironment has been known to play an integral role in tumor progression, the function of nonresident bone marrow-derived cells (BMDC) remains to be determined in neurologic tumors. Here we identified the contribution of BMDC recruitment in mediating malignant transformation from low- to high-grade gliomas.Experimental Design: We analyzed human blood and tumor samples from patients with low- and high-grade gliomas. A spontaneous platelet-derived growth factor (PDGF) murine glioma model (RCAS) was utilized to recapitulate human disease progression. Levels of CD11b+/GR1+ BMDCs were analyzed at discrete stages of tumor progression. Using bone marrow transplantation, we determined the unique influence of BMDCs in the transition from low- to high-grade glioma. The functional role of these BMDCs was then examined using a JAK 1/2 inhibitor (AZD1480).Results: CD11b+ myeloid cells were significantly increased during tumor progression in peripheral blood and tumors of glioma patients. Increases in CD11b+/GR1+ cells were observed in murine peripheral blood, bone marrow, and tumors during low-grade to high-grade transformation. Transient blockade of CD11b+ cell expansion using a JAK 1/2 Inhibitor (AZD1480) impaired mobilization of these cells and was associated with a reduction in tumor volume, maintenance of a low-grade tumor phenotype, and prolongation in survival.Conclusions: We demonstrate that impaired recruitment of CD11b+ myeloid cells with a JAK1/2 inhibitor inhibits glioma progression in vivo and prolongs survival in a murine glioma model. Clin Cancer Res; 23(12); 3109-19. ©2016 AACR.

Prophylactic orthosteric inhibition of leukocyte integrin CD11b/CD18 prevents long-term fibrotic kidney failure in cynomolgus monkeys.

Ischaemic acute kidney injury (AKI), an inflammatory disease process, often progresses to chronic kidney disease (CKD), with no available effective prophylaxis. This is in part due to lack of clinically relevant CKD models in non-human primates. Here we demonstrate that inhibition of the archetypal innate immune receptor CD11b/CD18 prevents progression of AKI to CKD in cynomolgus monkeys. Severe ischaemia-reperfusion injury of the right kidney, with subsequent periods of the left ureter ligation, causes irreversible right kidney failure 3, 6 or 9 months after AKI. Moreover, prophylactic inactivation of CD11b/CD18, using the orthosteric CD11b/CD18 inhibitor mAb107, improves microvascular perfusion and histopathology, reduces intrarenal pro-inflammatory mediators and salvages kidney function long term. These studies reveal an important early role of CD11b+ leukocytes in post-ischaemic kidney fibrosis and failure, and suggest a potential early therapeutic intervention to mitigate progression of ischaemic AKI to CKD in humans.

Treatment with the KCa3.1 inhibitor TRAM-34 during diabetic ketoacidosis reduces inflammatory changes in the brain.

BACKGROUND: Diabetic ketoacidosis (DKA) causes brain injuries in children ranging from subtle to life-threatening. Previous studies suggest that DKA-related brain injury may involve both stimulation of Na-K-Cl cotransport and microglial activation. Other studies implicate the Na-K-Cl cotransporter and the Ca-activated K channel KCa3.1 in activation of microglia and ischemia-induced brain edema. In this study, we determined whether inhibiting cerebral Na-K-Cl cotransport or KCa3.1 could reduce microglial activation and decrease DKA-related inflammatory changes in the brain. METHODS: Using immunohistochemistry, we investigated cellular alterations in brain specimens from juvenile rats with DKA before, during and after insulin and saline treatment. We compared findings in rats treated with and without bumetanide (an inhibitor of Na-K-Cl cotransport) or the KCa3.1 inhibitor TRAM-34. RESULTS: Glial fibrillary acidic protein (GFAP) staining intensity was increased in the hippocampus during DKA, suggesting reactive astrogliosis. OX42 staining intensity was increased during DKA in the hippocampus, cortex and striatum, indicating microglial activation. Treatment with TRAM-34 decreased both OX42 and GFAP intensity suggesting a decreased inflammatory response to DKA. Treatment with bumetanide did not significantly alter OX42 or GFAP intensity. CONCLUSIONS: Inhibiting KCa3.1 activity with TRAM-34 during DKA treatment decreases microglial activation and reduces reactive astrogliosis, suggesting a decreased inflammatory response.