Analysis of Innate Immune Signaling Networks

National Institute Of Allergy And Infectious Diseases

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Abstract

Antibiotics represent a fundamental cornerstone of modern medicine. In addition to their use in the treatment of potentially serious infections in otherwise healthy individuals, they have enabled the development of modern approaches in surgery, chemotherapy, and transplantation, which rely on the ability to control infections that occur in the context of compromised tissue barriers and immunologic defenses. The CDC estimates that in 2022, more than 235 million antibiotic prescriptions were dispensed from pharmacies in the US, representing an annual rate of ~7 prescriptions per 10 Americans. There are two broad mechanistic categories of antibiotic: bactericidal (cidal) drugs that directly kill bacteria, and bacteriostatic (static) drugs that inhibit bacterial growth. Although prior reports have found particular effects of specific antibiotics on a wide range of infection outcomes, the broader immunologic consequences of treating bacteria with these two antibiotic classes remain less clear. In FY25, using a murine peritonitis model, we observed a bacteriostatic treatment was more protective than a bactericidal treatment. To understand the mechanisms underlying this unexpected difference, we compared macrophage responses to bactericidal-killed bacteria or those growth-arrested by bacteriostatic antibiotics. We found that clinical Gram-negative bacterial isolates exposed to bactericidal drugs induced more proinflammatory cytokines than those treated with bacteriostatic agents. Data from Tlr4-/- macrophages and reporter cells showed that released LPS levels were comparable across antibiotic treatment types and thus not responsible for the bactericidal/bacteriostatic difference. By contrast, bacterial DNA – released only by bactericidal treatments – exacerbated inflammatory signaling through TLR9. In the absence of TLR9 signaling, the in vivo efficacy of bactericidal drug treatment was completely rescued. This demonstrates that antibiotics can act in important, indirect ways distinct from bacterial inhibition: such as causing treatment failure by releasing DNA that induces overwhelming, TLR9-dependent inflammation. These data establish a novel link between how an antibiotic affects bacterial physiology and subsequent pattern recognition receptor (PRR) engagement, which may have relevance for tailoring antibiotic treatments to consider both bacterial clearance and the resulting innate immune inflammatory profile. Metabolism is crucial in regulating the inflammatory immune response in macrophages. Inflammatory macrophages utilize metabolic pathways like glycolysis, glutaminolysis, fatty acid, and itaconic acid metabolism to control inflammatory cytokine production through transcriptional and epigenetic mechanisms. However, the impact of metabolic changes during Toll-like receptor (TLR) priming or inflammasome triggering on non-transcriptional processes, such as inflammasome assembly and pyroptotic interleukin-1β (IL-1β) release, is unclear. In FY25, using mass spectrometry-based dynamic metabolomic profiling in human U937 macrophages, we found that TLR priming induces an acute phase of limited metabolic reprogramming around 1 h after stimulation, followed by a more dramatic metabolic shift after 18 h. Consistent with published work, this later shift is characterized by enhancement of glycolysis via the glycolytic shunt, upregulation of the pentose phosphate pathway, and redirection of metabolite flow from the traditional TCA cycle into the itaconic acid shunt. We find that subsequent NLRP3 inflammasome triggering leads to a further substantial metabolic reprogramming. Specific inhibitors targeting the metabolic nodes enhanced during triggering indicate that pyroptotic IL-1β release occurs independently of mitochondrial control of inflammasome assembly. This metapyroptotic licensing mechanism promotes pyroptosis and is crucial for IL-1β release in macrophages from patients with neonatal-onset multisystem inflammatory disease (NOMID). In additional studies of inflammasome regulation, we have investigated regulators of the activation process that are specifically involved when the inflammasome is primed by a complex stimulus. We have previously described that under dual-TLR ligand priming conditions, the kinase IRAK1 has a specific role in forming protein clusters that recruit inflammasome components and facilitate downstream inflammasome responses. In FY25 we have further established that this function of IRAK1 leads to specific modulation of the key inflammasome effector ASC. In live cell imaging studies, we find that IRAK1 clustering in dual TLR-primed cells is a prerequisite for ASC-dependent inflammasome assembly. We are currently investigating regions of the IRAK1 protein that control this signaling process. These studies could uncover therapeutic approaches to alleviate inflammasome driven inflammation by targeting activity of the IRAK1 protein. In a further extension of our inflammasome research, we are considering how these pathways respond to fungal pathogens. In collaboration with NIAID colleagues, we find that certain inflammasome component-deficient mouse strains have increased susceptibility to select fungal strains. We are using genetic screening methods described in project AI001106 to investigate candidate host signaling proteins that facilitate the response to fungal challenge. This work is ongoing.