RNAi-based modulation of IFN-γ signaling in skin.

Aberrant activation of interferon (IFN)-γ signaling plays a key role in several autoimmune skin diseases, including lupus erythematosus, alopecia areata, vitiligo, and lichen planus. Here, we identify fully chemically modified small interfering RNAs (siRNAs) that silence the ligand binding chain of the IFN-γ receptor (IFNGR1), for the modulation of IFN-γ signaling. Conjugating these siRNAs to docosanoic acid (DCA) enables productive delivery to all major skin cell types local to the injection site, with a single dose of injection supporting effective IFNGR1 protein reduction for at least 1 month in mice. In an ex vivo model of IFN-γ signaling, DCA-siRNA efficiently inhibits the induction of IFN-γ-inducible chemokines, CXCL9 and CXCL10, in skin biopsies from the injection site. Our data demonstrate that DCA-siRNAs can be engineered for functional gene silencing in skin and establish a path toward siRNA treatment of autoimmune skin diseases.

Characterization of the Antigenic Heterogeneity of Lipoarabinomannan, the Major Surface Glycolipid of Mycobacterium tuberculosis, and Complexity of Antibody Specificities toward This Antigen.

Lipoarabinomannan (LAM), the major antigenic glycolipid of Mycobacterium tuberculosis, is an important immunodiagnostic target for detecting tuberculosis (TB) infection in HIV-1-coinfected patients, and is believed to mediate a number of functions that promote infection and disease development. To probe the human humoral response against LAM during TB infection, several novel LAM-specific human mAbs were molecularly cloned from memory B cells isolated from infected patients and grown in vitro. The fine epitope specificities of these Abs, along with those of a panel of previously described murine and phage-derived LAM-specific mAbs, were mapped using binding assays against LAM Ags from several mycobacterial species and a panel of synthetic glycans and glycoconjugates that represented diverse carbohydrate structures present in LAM. Multiple reactivity patterns were seen that differed in their specificity for LAM from different species, as well as in their dependence on arabinofuranoside branching and nature of capping at the nonreducing termini. Competition studies with mAbs and soluble glycans further defined these epitope specificities and guided the design of highly sensitive immunodetection assays capable of detecting LAM in urine of TB patients, even in the absence of HIV-1 coinfection. These results highlighted the complexity of the antigenic structure of LAM and the diversity of the natural Ab response against this target. The information and novel reagents described in this study will allow further optimization of diagnostic assays for LAM and may facilitate the development of potential immunotherapeutic approaches to inhibit the functional activities of specific structural motifs in LAM.

MicroRNA 122, Regulated by GRLH2, Protects Livers of Mice and Patients From Ethanol-Induced Liver Disease.

BACKGROUND & AIMS: Chronic, excessive alcohol consumption leads to alcoholic liver disease (ALD) characterized by steatosis, inflammation, and eventually cirrhosis. The hepatocyte specific microRNA 122 (MIR122) regulates hepatocyte differentiation and metabolism. We investigated whether an alcohol-induced decrease in level of MIR122 contributes to development of ALD. METHODS: We obtained liver samples from 12 patients with ALD and cirrhosis and 9 healthy individuals (controls) and analyzed them by histology and immunohistochemistry. C57Bl/6 mice were placed on a Lieber-DeCarli liquid diet, in which they were fed ethanol for 8 weeks, as a model of ALD, or a control diet. These mice were also given injections of CCl4, to increase liver fibrosis, for 8 weeks. On day 28, mice with ethanol-induced liver disease and advanced fibrosis, and controls, were given injections of recombinant adeno-associated virus 8 vector that expressed the primary miR-122 transcript (pri-MIR122, to overexpress MIR122 in hepatocytes) or vector (control). Two weeks before ethanol feeding, some mice were given injections of a vector that expressed an anti-MIR122, to knock down its expression. Serum and liver tissues were collected; hepatocytes and liver mononuclear cells were analyzed by histology, immunoblots, and confocal microscopy. We performed in silico analyses to identify targets of MIR122 and chromatin immunoprecipitation quantitative polymerase chain reaction analyses in Huh-7 cells. RESULTS: Levels of MIR122 were decreased in liver samples from patients with ALD and mice on the Lieber-DeCarli diet, compared with controls. Transgenic expression of MIR122 in hepatocytes of mice with ethanol-induced liver disease and advanced fibrosis significantly reduced serum levels of alanine aminotransferase (ALT) and liver steatosis and fibrosis, compared with mice given injections of the control vector. Ethanol feeding reduced expression of pri-MIR122 by increasing expression of the spliced form of the transcription factor grainyhead like transcription factor 2 (GRHL2) in liver tissues from mice. Levels of GRHL2 also were increased in liver tissues from patients with ALD, compared with controls; increases correlated with decreases in levels of MIR122 in human liver. Mice given injections of the anti-MIR122 before ethanol feeding had increased steatosis, inflammation, and serum levels of alanine aminotransferase compared with mice given a control vector. Levels of hypoxia-inducible factor 1 alpha (HIF1α) mRNA, a target of MIR122, were increased in liver tissues from patients and mice with ALD, compared with controls. Mice with hepatocyte-specific disruption of Hif1α developed less-severe liver injury following administration of ethanol, injection of anti-MIR122, or both. CONCLUSIONS: Levels of MIR122 decrease in livers from patients with ALD and mice with ethanol-induced liver disease, compared with controls. Transcription of MIR122 is inhibited by GRHL2, which is increased in livers of mice and patients with ALD. Expression of an anti-MIR122 worsened the severity of liver damage following ethanol feeding in mice. MIR122 appears to protect the liver from ethanol-induced damage by reducing levels of HIF1α. These processes might be manipulated to reduce the severity of ALD in patients.

Activation of antitumor immune responses by Ganoderma formosanum polysaccharides in tumor-bearing mice.

Fungi of the genus Ganoderma are basidiomycetes that have been used as traditional medicine in Asia and have been shown to exhibit various pharmacological activities. We recently found that PS-F2, a polysaccharide fraction purified from the submerged culture broth of Ganoderma formosanum, stimulates the maturation of dendritic cells and primes a T helper 1 (Th1)-polarized adaptive immune response in vivo. In this study, we investigated whether the immune adjuvant function of PS-F2 can stimulate antitumor immune responses in tumor-bearing mice. Continuous intraperitoneal or oral administration of PS-F2 effectively suppressed the growth of colon 26 (C26) adenocarcinoma, B16 melanoma, and sarcoma 180 (S180) tumor cells in mice without adverse effects on the animals' health. PS-F2 did not cause direct cytotoxicity on tumor cells, and it lost the antitumor effect in mice with severe combined immunodeficiency (SCID). CD4(+) T cells, CD8(+) T cells, and serum from PS-F2-treated tumor-bearing mice all exhibited antitumor activities when adoptively transferred to naïve animals, indicating that PS-F2 treatment stimulates tumor-specific cellular and humoral immune responses. These data demonstrate that continuous administration of G. formosanum polysaccharide PS-F2 can activate host immune responses against ongoing tumor growth, suggesting that PS-F2 can potentially be developed into a preventive/therapeutic agent for cancer immunotherapy.

A Keratinocyte-Tethered Biologic Enables Location-Precise Treatment in Mouse Vitiligo.

Despite the central role of IFN-γ in vitiligo pathogenesis, systemic IFN-γ neutralization is an impractical treatment option owing to strong immunosuppression. However, most patients with vitiligo present with <20% affected body surface area, which provides an opportunity for localized treatments that avoid systemic side effects. After identifying keratinocytes as key cells that amplify IFN-γ signaling during vitiligo, we hypothesized that tethering an IFN-γ‒neutralizing antibody to keratinocytes would limit anti‒IFN-γ effects on the treated skin for the localized treatment. To that end, we developed a bispecific antibody capable of blocking IFN-γ signaling while binding to desmoglein expressed by keratinocytes. We characterized the effect of the bispecific antibody in vitro, ex vivo, and in a mouse model of vitiligo. Single-photon emission computed tomography/computed tomography biodistribution and serum assays after local footpad injection revealed that the bispecific antibody had improved skin retention, faster elimination from the blood, and less systemic IFN-γ inhibition than the nontethered version. Furthermore, the bispecific antibody conferred localized protection almost exclusively to the treated footpad during vitiligo, which was not possible by local injection of the nontethered anti‒IFN-γ antibody. Thus, keratinocyte tethering proved effective while significantly diminishing the off-tissue effects of IFN-γ blockade, offering a safer treatment strategy for localized skin diseases, including vitiligo.