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Antimicrobials can impact bacterial physiology and host immunity with negative treatment outcomes. Extensive exposure to antifolate antibiotics promotes thymidine-dependent Staphylococcus aureus small colony variants (TD-SCVs), commonly associated with worse clinical outcomes. We show that antibiotic-mediated disruption of thymidine synthesis promotes elevated levels of the bacterial second messenger cyclic di-AMP (c-di-AMP), consequently inducing host STING activation and inflammation. An initial antibiotic screen in Firmicutes revealed that c-di-AMP production was largely driven by antifolate antibiotics targeting dihydrofolate reductase (DHFR), which promotes folate regeneration required for thymidine biosynthesis. Additionally, TD-SCVs exhibited excessive c-di-AMP production and STING activation in a thymidine-dependent manner. Murine lung infection with TD-SCVs revealed STING-dependent elevation of proinflammatory cytokines, causing higher airway neutrophil infiltration and activation compared with normal-colony S. aureus and hemin-dependent SCVs. Collectively, our results suggest that thymidine metabolism disruption in Firmicutes leads to elevated c-di-AMP-mediated STING-dependent inflammation, with potential impacts on antibiotic usage and infection outcomes.

SummaryAntibiotics remain one of the most effective methods for controlling bacterial infection. However, the diverse impacts of antimicrobials on bacterial physiology and host immunity remain unclear. A comprehensive antibiotic screen revealed that disruption of thymidine synthesis in Firmicutes with anti-folate antibiotics promoted elevated levels of the bacterial second messenger cyclic di-AMP, and consequently induced host STING activation during infection. Extensive exposure to antibiotics targeting folate synthesis drives the emergence of thymidine-dependent Staphylococcus aureus SCVs (TD-SCVs). Respiratory infections with TD-SCVs are common among children with cystic fibrosis and are associated with worse clinical outcomes, although the underlying pathophysiological mechanisms remain to be defined. Our study reveals that TD-SCV isolates exhibited excessive c-di-AMP production and STING activation in a thymidine-dependent manner. Murine lung infection with TD-SCVs revealed STING-dependent elevation of proinflammatory cytokines, leading to higher airway neutrophil infiltration and activation comparing to normal colony S. aureus and hemin-dependent SCV. Our results suggest the elevated inflammatory capacity of TD-SCVs contribute to their pathogenesis and revealed a new aspect of STING signaling in the airway by characterizing its role in neutrophil recruitment.

Bacterial and host cyclic dinucleotides (cdNs) mediate cytosolic immune responses through the STING signaling pathway, although evidence suggests that alternative pathways exist. We used cdN-conjugated beads to biochemically isolate host receptors for bacterial cdNs, and we identified the oxidoreductase RECON. High-affinity cdN binding inhibited RECON enzyme activity by simultaneously blocking the substrate and cosubstrate sites, as revealed by structural analyses. During bacterial infection of macrophages, RECON antagonized STING activation by acting as a molecular sink for cdNs. Bacterial infection of hepatocytes, which do not express STING, revealed that RECON negatively regulates NF-κB activation. Loss of RECON activity, via genetic ablation or inhibition by cdNs, increased NF-κB activation and reduced bacterial survival, suggesting that cdN inhibition of RECON promotes a proinflammatory, antibacterial state that is distinct from the antiviral state associated with STING activation. Thus, RECON functions as a cytosolic sensor for bacterial cdNs, shaping inflammatory gene activation via its effects on STING and NF-κB.

Background: Despite significant clinical benefit of checkpoint inhibitors (CPI) in some settings, unfortunately, the majority of patients still fail to respond and/or develop resistance. This is likely due to a complex set of factors including, but not limited to, the presence of immunosuppressive myeloid cells and/or T cell exhaustion due to chronic TCR activation in the absence of sufficient costimulation. ALPN-202, a variant CD80 vIgD™-Fc fusion protein that mediates PD-L1-dependent CD28 costimulation and blocks PD-L1 and CTLA-4, was designed to overcome several of these suppressive mechanisms. The objective of these studies was to measure effects of ALPN-202 on the suppression of T cell activation by M2c macrophages and to determine in vivo effects of ALPN-202 on T cell exhaustion in an adoptively transferred human TCR transgenic tumor model. Methods: M2c macrophages differentiated from primary monocytes with M-CSF and IL-10 were cocultured for 72hr with T cells, anti-CD3 and a titration of ALPN-202, anti-PD-1, anti-PD-L1, or anti-CTLA-4 antibodies. Cytokine concentrations in the culture media were measured at 24 and 72 hrs, and T cells and macrophages characterized at 72 hrs by flow cytometry. To measure its anti-tumor activity and effect on T cell exhaustion in vivo, ALPN-202 was evaluated in a humanized model using anti-HPV E6 TCR-transduced human T cells transferred into immunodeficient NSG mice bearing HPV+ SCC152 squamous cell tumors stably expressing PD-L1. Tumor volume was measured twice weekly and on day 38 tumors were harvested, digested, and intratumoral T cells characterized for expression of exhaustion markers. Results: In the in vitro coculture assay, ALPN-202 increased T cell proliferation and production of IL-2, IFNγ, TNFα, GM-CSF, IL-6, and IL-21 significantly more potently than CPI alone. Additionally, the M2c macrophages in the presence of ALPN-202 displayed a dose-dependent elevation of MHC II, CD80, and CD86, indicative of a more pro-inflammatory, M1-like phenotype. In the SCC152 tumor model, ALPN-202 induced a more robust anti-tumor response than CPI while the intratumoral T cells expressed lower levels of exhaustion markers. Conclusions: As a dual checkpoint inhibitor and conditional CD28-costimulator, ALPN-202 induced robust and selective T cell costimulation in vitro which overcame M2c macrophage-mediated suppression more potently than CPI alone. Intriguingly, not only were T cells vigorously activated, but M2c macrophages transitioned to a more M1-like proinflammatory phenotype. In a humanized tumor model, ALPN-202 treatment resulted in potent anti-tumor activity with reduced signs of T cell exhaustion in the TME. The data suggest that by combining CD28 costimulation with CPI, ALPN-202 may provide a more robust and persistent anti-tumor T cell response compared to CPI alone. Citation Format: Mark F. Maurer, Siddarth Chandrasekaran, Katherine Lewis, Sherri Mudri, Kayla Kleist, Fariha Ahmed-Qadri, Chelsea Gudgeon, Steven D. Levin, Stacey R. Dillon, Kristine M. Swiderek, Stanford L. Peng. ALPN-202 combines checkpoint inhibition with conditional T cell costimulation to overcome T cell suppression by M2c macrophages and improve the durability of engineered T cell anti-tumor responses [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-085.

Host cells have evolved a sophisticated arsenal of germ-line encoded pattern recognition receptors that detect a vast array of microorganisms in distinct tissues and cellular compartments. Cytoplasmic sensors monitor for microbes that gain access to the host cell cytosol following breach of the plasma membrane or intracellular invasion. This sensing is achieved through detection of invariant molecular patterns associated with microorganisms or cell stress responses that indicate the presence of a pathogen. Cyclic dinucleotides (cdNs) of both bacterial and host origin have emerged as important molecules that are sensed during infection. Details about the receptors cdNs engage in host cells and the responses those interactions elicit are just coming to light. We have recently discovered the first bacterial cdN-specific pattern recognition receptor, the oxidoreductase RECON. Our work on this receptor has revealed its central role in directing innate immune responses when engaged by bacterial cdNs. These findings reveal a new pattern recognition receptor specific for bacterial cdNs that orchestrates cytosolic immune surveillance by shaping downstream inflammatory gene activation.