Intranasal administration of nucleus-deliverable GATA3-TMD alleviates the symptoms of allergic asthma.

Although the T helper 2 (Th2) subset is a critical player in the humoral immune response to extracellular parasites and suppression of Th1-mediated inflammation, Th2 cells have been implicated in allergic inflammatory diseases such as asthma, allergic rhinitis, and atopic dermatitis. GATA binding protein 3 (GATA3) is a primary transcription factor that mediates Th2 differentiation and secretion of Th2 cytokines, including IL-4, IL-5, and IL-13. Here, a nucleus-deliverable form of GATA3-transcription modulation domain (TMD) (ndG3-TMD) was generated using Hph-1 human protein transduction domain (PTD) to modulate the transcriptional function of endogenous GATA3 without genetic manipulation. ndG3-TMD was shown to be efficiently delivered into the cell nucleus quickly without affecting cell viability or intracellular signaling events for T cell activation. ndG3-TMD exhibited a specific inhibitory function for the endogenous GATA3-mediated transcription, such as Th2 cell differentiation and Th2-type cytokine production. Intranasal administration of ndG3-TMD significantly alleviated airway hyperresponsiveness, infiltration of immune cells, and serum IgE level in an OVA-induced mouse model of asthma. Also, Th2 cytokine secretion by the splenocytes isolated from the ndG3-TMD-treated mice substantially decreased. Our results suggest that ndG3-TMD can be a new therapeutic reagent to suppress Th2-mediated allergic diseases through intranasal delivery.

Intranuclear Delivery of Nuclear Factor-Kappa B p65 in a Rat Model of Tooth Replantation.

After avulsion and replantation, teeth are at risk of bone and root resorption. The present study aimed to demonstrate that the intra-nuclear transducible form of transcription modulation domain of p65 (nt-p65-TMD) can suppress osteoclast differentiation in vitro, and reduce bone resorption in a rat model of tooth replantation. Cell viability and nitric oxide release were evaluated in RAW264.7 cells using CCK-8 assay and Griess reaction kit. Osteoclast differentiation was evaluated using quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and tartrate-resistant acid phosphatase (TRAP) staining. Thirty-two maxillary rat molars were extracted and stored in saline (n = 10) or 10 µM nt-p65-TMD solution (n = 22) before replantation. After 4 weeks, specimens were scored according to the inflammatory pattern using micro-computed tomography (CT) imaging and histological analyses. nt-p65-TMD treatment resulted in significant reduction of nitric oxide release and osteoclast differentiation as studied using PCR and TRAP staining. Further, micro-CT analysis revealed a significant decrease in bone resorption in the nt-p65-TMD treatment group (p < 0.05). Histological analysis of nt-p65-TMD treatment group showed that not only bone and root resorption, but also inflammation of the periodontal ligament and epithelial insertion was significantly reduced. These findings suggest that nt-p65-TMD has the unique capabilities of regulating bone remodeling after tooth replantation.

Intranuclear interactomic inhibition of NF-κB suppresses LPS-induced severe sepsis.

Suppression of nuclear factor-κB (NF-κB) activation, which is best known as a major regulator of innate and adaptive immune responses, is a potent strategy for the treatment of endotoxic sepsis. To inhibit NF-κB functions, we designed the intra-nuclear transducible form of transcription modulation domain (TMD) of RelA (p65), called nt-p65-TMD, which can be delivered effectively into the nucleus without influencing the cell viability, and work as interactomic inhibitors via disruption of the endogenous p65-mediated transcription complex. nt-p65-TMD effectively inhibited the secretion of pro-inflammatory cytokines, including TNF-α, IL-1ß, or IL-6 from BV2 microglia cells stimulated by lipopolysaccharide (LPS). nt-p65-TMD did not inhibit tyrosine phosphorylation of signaling mediators such as ZAP-70, p38, JNK, or ERK involved in T cell activation, but was capable of suppressing the transcriptional activity of NF-κB without the functional effect on that of NFAT upon T-cell receptor (TCR) stimulation. The transduced nt-p65-TMD in T cell did not affect the expression of CD69, however significantly inhibited the secretion of T cell-specific cytokines such as IL-2, IFN-γ, IL-4, IL-17A, or IL-10. Systemic administration of nt-p65-TMD showed a significant therapeutic effect on LPS-induced sepsis model by inhibiting pro-inflammatory cytokines secretion. Therefore, nt-p65-TMD can be a novel therapeutics for the treatment of various inflammatory diseases, including sepsis, where a transcription factor has a key role in pathogenesis, and further allows us to discover new functions of p65 under normal physiological condition without genetic alteration.

Lrig1-expression confers suppressive function to CD4+ cells and is essential for averting autoimmunity via the Smad2/3/Foxp3 axis.

Regulatory T cells (Treg) are CD4+ T cells with immune-suppressive function, which is defined by Foxp3 expression. However, the molecular determinants defining the suppressive population of T cells have yet to be discovered. Here we report that the cell surface protein Lrig1 is enriched in suppressive T cells and controls their suppressive behaviors. Within CD4+ T cells, Treg cells express the highest levels of Lrig1, and the expression level is further increasing with activation. The Lrig1+ subpopulation from T helper (Th) 17 cells showed higher suppressive activity than the Lrig1- subpopulation. Lrig1-deficiency impairs the suppressive function of Treg cells, while Lrig1-deficient naïve T cells normally differentiate into other T cell subsets. Adoptive transfer of CD4+Lrig1+ T cells alleviates autoimmune symptoms in colitis and lupus nephritis mouse models. A monoclonal anti-Lrig1 antibody significantly improves the symptoms of experimental autoimmune encephalomyelitis. In conclusion, Lrig1 is an important regulator of suppressive T cell function and an exploitable target for treating autoimmune conditions.

Interactomic inhibition of Eomes in the nucleus alleviates EAE via blocking the conversion of Th17 cells into non-classic Th1 cells.

Th17 cells are implicated in the pathogenesis of several autoimmune diseases. During the inflammation, Th17 cells exposed to IL-12 can shift towards the Th1 phenotype. These shifted cells are defined as 'non-classic Th1 cells'. Th17-derived non-classic Th1 cells play a critical role in late-onset chronic inflammatory diseases and are more pathogenic than the unshifted Th17 cells. Eomes is a transcription factor highly expressed in non-classic Th1 cells. To study the functional role of Eomes without genetic alteration, novel recombinant protein, ntEomes-TMD, was generated by fusing TMD of Eomes and Hph-1-PTD that facilitate intracellular delivery of its cargo molecule. ntEomes-TMD was delivered into the nucleus of the cells without influencing the T cell activation and cytotoxicity. ntEomes-TMD specifically inhibited the Eomes- and ROR-γt-mediated transcription and suppressed the Th1 and Th17 differentiation. Interestingly, ntEomes-TMD blocked the generation of non-classic Th1 cells from Th17 cells, leading to the inhibition of IFN-γ and GM-CSF secretion. In EAE, ntEomes-TMD alleviated the symptoms of EAE, and the combination treatment using ntEomes-TMD and anti-IL-17 mAb together showed better therapeutic efficacy than anti-IL-17 mAb treatment. The results suggest that ntEomes-TMD can be a new therapeutic reagent for treating chronic inflammatory diseases associated with non-classic Th1 cells.