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Diffuse midline glioma (DMG) is a high-grade glioma with a median overall survival of 9-11 months. Radiotherapy is the only recognized treatment. The DMG tumor microenvironment (TME) contains few, if any, tumor infiltrating lymphocytes (TILs) or inflammatory cytokines, thus is distinctive of an ‘immunologically cold’ tumor/TME.1 DMG lack the expression of immunosuppressive immune checkpoint proteins, likely explaining the failure of immune checkpoint inhibitors (ICI) tested under clinical trials for DMG patients, and suggestive of an alternative mechanism underpinning the cold TME. 1 Glioblastomas also harbor a cold TME, which can be somewhat explained by T cell lymphopenia, influenced by the sequestration of T cells in the bone marrow (through Beta-arrestin-induced internalization of Sphingosine-1-phosphate receptor 1 [S1PR1]). 2 Dopaminergic activation of Beta-arrestin and hence S1PR1 internalization, is potentially regulated through dopaminergic peripheral nerves in primary and secondary lymphoid organs, regulated by the Dopamine receptor D2 (DRD2), that is highly expressed on T cells. ONC201 is a potent DRD2 antagonist, currently in phase I-III clinical trials for DMG patients, alone and in combination with radiotherapy and the PI3K/AKT inhibitor paxalisib (NCT05009992). Proteomic profiling of DMG patient-derived cells +/-ONC201 showed increased expression of several antigen presenting pathway proteins, including Beta-2-microglobulin (B2M) and HLA class I histocompatibility antigen, A alpha chain (HLA-A). This was confirmed in vivo using SU-DIPG-VI patient-derived xenograft mouse model tissues +/-ONC201 alone, and together with paxalisib. Excitingly, this combination (given orally) promoted the recruitment of TILs to the tumor, revealing novel immunomodulatory effects. In vivo, ONC201 promoted the expression of EMILIN-3, a TGF-β antagonist that is known to inhibit HLA-A/B2M expression, possibly explaining the increased MHC-I activity. This study uncovers a novel link between treatment of DMG with ONC201 and paxalisib and the role dopaminergic peripheral nerves signaling may play on the sequestration of T cells within lymphoid organs and lymphopenia.

Background Steroid-insensitive endotypes of asthma are an important clinical problem requiring effective treatments. They are associated with non-eosinophilic inflammatory responses and bacterial infections. Macrolide therapy is effective in steroid-insensitive endotypes, such as non-eosinophilic asthma, however the mechanisms of how they work is unknown. Objectives To determine the efficacy of macrolide and non-macrolide antibiotic treatments in infection-induced, severe, steroid-insensitive allergic airways disease (SSIAAD). Methods Mouse models of Chlamydia and Haemophilus lung infection-induced SSIAAD were used to investigate the effects of clarithromycin and amoxicillin treatment on immune responses and AHR in steroid-sensitive AAD and SSIAAD compared to dexamethasone treatment. Results Amoxicillin and clarithromycin had similar anti-microbial effects on infection. Amoxicillin did not suppress either form of AAD, but restored steroid sensitivity in SSIAAD by reducing infection. In contrast, clarithromycin alone widely suppressed inflammation and AHR in both steroid-sensitive and SSIAAD. This occurred through reductions in both Th2 responses that drive steroid-sensitive, eosinophilic AAD, and TNF-α and IL-17 responses that induce neutrophilic SSIAAD. Conclusions Macrolides have broad anti-inflammatory effects that are independent of their anti-microbial effects. The specific responses suppressed are dependent upon the responses that dominate during disease

BACKGROUND: Severe, steroid-insensitive (SSI) asthma is a substantial clinical problem. Effective treatments are urgently required, however, their development is hampered by a lack of understanding of the mechanisms that promote disease. SSI asthma is associated with respiratory infections and non-eosinophilic endotypes of disease, including neutrophilic asthma. OBJECTIVES: To develop and use mouse models of SSI neutrophilic asthma to investigate pathogenic mechanisms involving microRNA (miR)-21, phosphoinositide-3-kinase (PI3K) and histone deacetylase (HDAC)2 in order to identify new therapeutic approaches. METHODS: Novel mouse models of respiratory infection and ovalbumin-induced, SSI neutrophilic allergic airway disease (SSIAAD) in BALB/c mice were developed. The roles of infection-induced miR-21 expression and PI3K-dependent signalling in the lung were examined using a specific miR-21 inhibitor (antagomir-21) and the pan-PI3K inhibitor LY294002. RESULTS: Infection induced a miR-21-dependent, PI3K-mediated signalling pathway that decreased nuclear HDAC2 levels and promoted steroid-insensitive neutrophilic inflammation and airway hyper-responsiveness (AHR) in AAD. Inhibition of miR-21 or PI3K suppressed nuclear pAkt levels and restored HDAC2 levels and steroid sensitivity. CONCLUSIONS: We have identified a novel role for a miR-21/PI3K/HDAC2 signalling axis in SSIAAD. Our data highlights miR-21 as a novel target for treating this form of asthma.

Chlamydia trachomatis is the most common bacterial STI and frequently results in reproductive tract (RT) sequelae such as pelvic inflammatory disease and infertility. However, the immune processes involved in the clearance and immunopathology of Chlamydia infection are not well understood. In previous studies we showed that IFN-ε, a novel type I IFN that is exclusively and constitutively expressed in the female RT, plays an important role in protecting against Chlamydia infections. Here, we examined the effects of IFN-ɛ on cellular responses in the female RT in order to elucidate the mechanisms by which IFN-ɛ protects against Chlamydia infections. Female WT and IFN-ε-/- C57BL/6 mice were pre-treated with progesterone and infected intra-vaginally with Chlamydia muridarum or sham-infected. Uterine horns were harvested and the effects of IFN-ε deficiency on Chlamydia infection, immune factor expression and cellular infiltration were assessed using real-time qPCR and flow cytometry. We show that IFN-ε-/- mice have increased Chlamydia 16S expression in the upper RT which correlated with fewer NK cells and tissue-resident uterine NK cells at 3 days post infection. IFN-γ+ CD45+ cells were also decreased in the infected IFN-ε-/- mice, of which over 60% were NK cells. These changes were associated with reduced iNOS and STAT1 expression. These findings suggest that IFN-ε may protect against Chlamydia RT infections by potentiating the recruitment of protective IFN-γ-producing NK cells.

Cigarette smoke-induced chronic obstructive pulmonary disease (COPD) is a life-threatening inflammatory disorder of the lung. The development of effective therapies for COPD has been hampered by the lack of an animal model that mimics the human disease in a short time-frame.We have created a mouse model of cigarette smoke-induced COPD that develops the hallmark features of the human condition in a short amount of time. Tightly controlled amounts of cigarette smoke were delivered to the airways of mice, and the development of the pathological features of COPD were assessed. The roles of macrophages and mast cell (MC) tryptase in pathogenesis were evaluated using depletion and in vitro studies and MC protease-6 deficient mice. After 8 weeks of smoke exposure, wild-type mice developed chronic inflammation, mucus hypersecretion, airway remodeling, emphysema, and reduced lung function. These characteristic features of COPD were glucocorticoid-resistant and did not spontaneously resolve. Systemic effects on skeletal muscle and the heart, and increased susceptibility to respiratory infections also were observed. We demonstrate here that macrophages and tryptase-expressing MCs were required for the development of COPD. Recombinant MC tryptase induced pro-inflammatory responses from cultured macrophages. This model can be used to better understand multiple aspects of COPD pathogenesis.

We have previously shown that chlamydial lung infections in early-life drive the development of chronic airway hyper-responsiveness (AHR). To elucidate the mechanisms involved, we used a murine model of chlamydial lung infection, to investigate the influence of IL-13 on infection and infection-induced AHR. Infected IL-13-/- mice had less severe infection, reduced inflammation (neutrophils, dendritic cells, macrophages and T cells), less mucus secreting cells (MSCs) in the airways and attenuated AHR. More severe infection in wild-type (WT) mice was associated with the development of alternatively activated macrophages (increased Arg-1, FIZZ-1 and Ym-1), which were suppressed in IL-13-/- mice. Surprisingly, infection of WT mice did not induce any detectable increase in IL-13 expression. However, infection decreased the level of the IL-13 decoy receptor (IL-13Rα2), which may allow constitutive IL-13 to signal. Interestingly, reconstitution of IL-13 in IL-13-/- mice restored infection-induced inflammation and MSC formation, but not AHR. Furthermore, infection-induced inflammation and mucus were Stat-6-independent, whilst infection-induced AHR was dependent upon Stat-6. Thus, early-life respiratory chlamydial infection results in decreased decoy receptor expression, which allows constitutive IL-13 to signal and promote more severe infection, which drives the development of chronic AHR that persists into adulthood.