The dynamic changes of RNA N6-methyl-adenosine (m6A) during cancer progression contribute to quick adaption to microenvironmental changes. Here, we profiled the cancer cell m6A dynamics in the hypoxic tumor niche and its pathological consequences in glioblastoma multiforme (GBM). The m6A demethylase ALKBH5 was induced in GBM models under hypoxic conditions and was associated with a hypoxic gene signature in GBM patient samples. Depletion or inactivation of ALKBH5 in GBM cells significantly suppressed hypoxia-induced tumor-associated macrophage (TAM) recruitment and immunosuppression in allograft tumors. Expression and secretion of CXCL8/IL8 were significantly suppressed in ALKBH5-deficient tumors. However, ALKBH5 did not regulate CXCL8 m6A directly. Instead, hypoxia-induced ALKBH5 erased m6A deposition from the lncRNA NEAT1, stabilizing the transcript and facilitating NEAT1-mediated paraspeckle assembly, which led to relocation of the transcriptional repressor SFPQ from the CXCL8 promoter to paraspeckles and, ultimately, upregulation of CXCL8/IL8 expression. Accordingly, ectopic expression of CXCL8 in ALKBH5-deficient GBM cells partially restored TAM recruitment and tumor progression. Together, this study links hypoxia-induced epitranscriptomic changes to the emergence of an immunosuppressive microenvironment facilitating tumor evasion. Significance: Hypoxia induces tumor immune microenvironment remodeling through an ALKBH5-mediated epigenetic and epitranscriptomic mechanism, providing potential immunotherapeutic strategies for treating glioblastoma.

SummaryInflammaging plays an important role in most age‐related diseases. However, the mechanism of inflammaging is largely unknown, and therapeutic control of inflammaging is challenging. Human alpha‐1 antitrypsin (hAAT) has immune‐regulatory, anti‐inflammatory, and cytoprotective properties as demonstrated in several disease models including type 1 diabetes, arthritis, lupus, osteoporosis, and stroke. To test the potential anti‐inflammaging effect of hAAT, we generated transgenic Drosophila lines expressing hAAT. Surprisingly, the lifespan of hAAT‐expressing lines was significantly longer than that of genetically matched controls. To understand the mechanism underlying the anti‐aging effect of hAAT, we monitored the expression of aging‐associated genes and found that aging‐induced expressions of Relish (NF‐ĸB orthologue) and Diptericin were significantly lower in hAAT lines than in control lines. RNA‐seq analysis revealed that innate immunity genes regulated by NF‐kB were significantly and specifically inhibited in hAAT transgenic Drosophila lines. To confirm this anti‐inflammaging effect in human cells, we treated X‐ray‐induced senescence cells with hAAT and showed that hAAT treatment significantly decreased the expression and maturation of IL‐6 and IL‐8, two major factors of senescence‐associated secretory phenotype. Consistent with results from Drosophila, RNA‐seq analysis also showed that hAAT treatment significantly inhibited inflammation related genes and pathways. Together, our results demonstrated that hAAT significantly inhibited inflammaging in both Drosophila and human cell models. As hAAT is a FDA‐approved drug with a confirmed safety profile, this novel therapeutic potential may make hAAT a promising candidate to combat aging and aging‐related diseases.

Heat shock or stress proteins (Hsps) are a group of proteins induced by a large number of xenobiotics, many of which are common in the working and living environment. The biological significance of the presence of antibodies against Hsps in humans is presently unknown. In the present study, 112 workers were selected and divided into four groups on the basis of their level of occupational exposure to benzene: a control group, two groups of workers exposed to either low (< 300 mg/m3) or high concentrations of benzene (> 300 mg/m3) and a group of workers who had experienced benzene poisoning. Blood samples from these workers were assayed for the number of peripheral white blood cells, concentration of hemoglobin, activities of serum superoxide dismutase (SOD), lymphocyte DNA damage and finally for the presence of antibodies to different human heat-shock proteins (Hsp27, Hsp60, Hsp71 and Hsp90). Benzene-poisoned workers showed a high incidence of antibodies against Hsp71 (approximately 40%) which was associated with a decrease in white blood cells (3.84+/-1.13 x 10(9)versus 7.68+/-1.84 x 10(9) in controls) and with an increase in activities of serum SOD (138.43+/-23.15 micro/ml) and lymphocyte DNA damage (18.7%). These data suggest that antibodies against Hsps can potentially be useful biomonitors to assess if workers are experiencing or have experienced abnormal xenobiotic-induced stress within their living and working environment.

Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease characterized by high levels of pathogenic autoantibodies and tissue damage. Multiple studies showed that dendritic cell (DC) activation plays a critical role in SLE pathogenesis. Human alpha 1 antitrypsin (hAAT) is a serine proteinase inhibitor with potent anti-inflammatory and cytoprotective properties. In this study, we first examined the effects of hAAT on the functions of DCs from lupus-prone mice, and we showed that hAAT treatment efficiently inhibited CpG- (TLR9 agonist) induced activation of bone marrow-derived conventional and plasmacytoid DCs as well as the production of pro-inflammatory cytokines. The hAAT treatment also attenuated DC help for B cell proliferation and immunoglobulin M (IgM) production. We next tested the protective effect of hAAT protein and gene therapy using recombinant adeno-associated virus 8 (rAAV8-CB-hAAT) in a spontaneous lupus mouse model, and we showed that both treatments decreased autoantibody levels. Importantly, rAAV8-CB-hAAT did not induce an immune response to its transgene product (hAAT), but it showed more pronounced therapeutic effects in reducing urine protein levels and extending the lifespan of these mice. These results indicate that AAT has therapeutic potential in the treatment of SLE in humans.

Diffuse alveolar hemorrhage (DAH) is a fatal complication in patients with lupus. DAH can be induced in B6 mice by an intraperitoneal injection of pristane. Since human alpha-1-antitrypsin (hAAT) is an anti-inflammatory and immuno-regulatory protein, we investigated the protective effect of hAAT against pristane-induced DAH in B6 mice and hAAT transgenic (hAAT-Tg) mice. We first showed that hAAT Tg expression lowers TNF-α production in B cells, as well as CD4+ T cells in untreated mice. Conversely, the frequency of regulatory CD4+CD25+ and CD4+CD25-IL-10+ cells was significantly higher in hAAT-Tg than in B6 mice. This confirmed the anti-inflammatory effect of hAAT that was observed even at steady state. One week after a pristane injection, the frequency of peritoneal Ly6Chi inflammatory monocytes and neutrophils in hAAT-Tg mice was significantly lower than that in B6 mice. Importantly, pristane-induced DAH was completely prevented in hAAT-Tg mice and this was associated with a modulation of anti- to pro-inflammatory myeloid cell ratio/balance. We also showed that treatment with hAAT decreased the severity of DAH in B6 mice. These results showed for the first time that hAAT has a therapeutic potential for the treatment of DAH.