Inflammatory response plays an essential role in the resolution of infections. However, inflammation can be detrimental to an organism and cause irreparable damage. For example, during sepsis, a cytokine storm can lead to multiple organ failures and often results in death. One of the strongest triggers of the inflammatory response is bacterial lipopolysaccharides (LPS), acting mostly through Toll-like receptor 4 (TLR4). Paradoxically, while exposure to LPS triggers a robust inflammatory response, repeated or prolonged exposure to LPS can induce a state of endotoxin tolerance, a phenomenon where macrophages and monocytes do not respond to new endotoxin challenges, and it is often associated with secondary infections and negative outcomes. The cellular mechanisms regulating this phenomenon remain elusive. We used metabolic measurements to confirm differences in the cellular metabolism of naïve macrophages and that of macrophages responding to LPS stimulation or those in the LPS-tolerant state. In parallel, we performed an unbiased secretome survey using quantitative mass spectrometry during the induction of LPS tolerance, creating the first comprehensive secretome profile of endotoxin-tolerant cells. The secretome changes confirmed that LPS-tolerant macrophages have significantly decreased cellular metabolism and that the proteins secreted by LPS-tolerant macrophages have a strong association with cell survival, protein metabolism, and the metabolism of reactive oxygen species.

Burkholderia pseudomallei is an environmental bacterium that causes melioidosis, a major community-acquired infection in tropical regions. Melioidosis presents with a range of clinical symptoms, is often characterized by a robust inflammatory response, may relapse after treatment, and results in high mortality rates. Lipopolysaccharide (LPS) of B. pseudomallei is a potent immunostimulatory molecule comprised of lipid A, core, and O-polysaccharide (OPS) components. Four B. pseudomallei LPS types have been described based on SDS-PAGE patterns that represent the difference of OPS–type A, type B, type B₂ and rough LPS. The majority of B. pseudomallei isolates are type A. We used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) followed by electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-QqTOF MS) and gas chromatography to characterize the lipid A of B. pseudomallei within LPS type A isolates. We determined that B. pseudomallei lipid A is represented by penta- and tetra-acylated species modified with 4-amino-4-deoxy-arabinose (Ara4N). The MALDI-TOF profiles from 171 clinical B. pseudomallei isolates, including 68 paired primary and relapse isolates and 35 within-host isolates were similar. We did not observe lipid A structural changes when the bacteria were cultured in different growth conditions. Dose-dependent NF-κB activation in HEK cells expressing TLR4 was observed using multiple heat-killed B. pseudomallei isolates and corresponding purified LPS. We demonstrated that TLR4-dependent NF-κB activation induced by heat-killed bacteria or LPS prepared from OPS deficient mutant was significantly greater than those induced by wild type B. pseudomallei. These findings suggest that the structure of B. pseudomallei lipid A is highly conserved in a wide variety of clinical and environmental circumstances but that the presence of OPS may modulate LPS-driven innate immune responses in melioidosis. Melioidosis is a tropical infection caused by Burkholderia pseudomallei. Disease ranges from mild to lethal, and patients who die from melioidosis have greater inflammatory responses than survivors. Identifying variation in bacterial structures that alter the immune response is therefore important. B. pseudomallei expresses lipopolysaccharide, a major stimulator of the immune response. Studies of other bacteria suggest that differences in the lipid A component of lipopolysaccharide result in variable stimulation of the immune system. In this study, we analyzed the chemical structure of lipid A of 171 clinical B. pseudomallei isolates, structural changes caused by different growth conditions, and innate immune responses induced. Surprisingly, we did not observe any variation of lipid A structure or different innate immune activation from different B. pseudomallei isolates. Our data suggest that lipid A structure of B. pseudomallei is highly conserved and that different inflammatory responses in patients may be caused by other factors.

Eosinophilic esophagitis (EoE) is a chronic gastrointestinal disorder characterized by food antigen–driven eosinophilic inflammation and hyperproliferation of esophageal mucosa. By utilizing a large-scale, proteomic screen of esophageal biopsies, we aimed to uncover molecular drivers of the disease. Proteomic analysis by liquid chromatography–tandem mass spectrometry identified 402 differentially expressed proteins (DEPs) that correlated with the EoE transcriptome. Immune cell–related proteins were among the most highly upregulated DEPs in EoE compared with controls, whereas proteins linked to epithelial differentiation were primarily downregulated. Notably, in the inflamed esophageal tissue, all 6 subunits of the minichromosome maintenance (MCM) complex, a DNA helicase essential for genomic DNA replication, were significantly upregulated at the gene and protein levels. Furthermore, treating esophageal epithelial cells with a known inhibitor of the MCM complex (ciprofloxacin) blocked esophageal epithelial proliferation. In a murine model of EoE driven by overexpression of IL-13, ciprofloxacin treatment decreased basal zone thickness and reduced dilated intercellular spaces by blocking the transition of epithelial cells through the S-phase of the cell cycle. Collectively, a broad-spectrum proteomic screen has identified the involvement of the MCM complex in EoE and has highlighted MCM inhibitors as potential therapeutic agents for the disease.

Macrophages harness pattern recognition receptors (PRRs) to detect conserved bacterial components and mount effective immune responses. Many Gram-negative bacteria modify their lipid A structures to limit recognition by Toll-like receptor 4 (TLR4) and cytosolic Caspase-11 lipopolysaccharide sensors. One common evasion strategy is to reduce the lipid A acylation state from hexa- to tetra-acylation. This alteration can limit binding to receptors and dampen subsequent immune signaling responses, yet the proteomic alterations associated with this altered immunogenicity remain incompletely understood. Here, we systematically profiled proteomic alterations induced by extracellular or transfected hexa-acylated Kdo2-lipid A (Kdo2) and tetra-acylated lipid-IVa (IVa) to assess TLR4-dependent, TLR4-independent, and non-canonical inflammasome activation pathways. Kdo2 elicited stronger inflammatory responses in immortalized bone-marrow-derived macrophages (iBMDMs), as evidenced by robust TNF production, Caspase-11 cleavage, and IL-1α/IL-1β release. In contrast, IVa elicited minimal TNF secretion and failed to effectively induce non-canonical inflammasome activation. Global label-free quantitative proteomic analysis of iBMDMs stimulated with a low dose of immunogenic LPS displayed route-specific immune signatures: enrichment of TNF signaling, interferon-associated pathways, and mitochondrial metabolic remodeling. Equimolar amounts of low-acylated LPS failed to effectively induce these immune signatures, supporting a threshold-dependent model in which the lipid A structure and route of exposure define inflammatory progression. Collectively, our findings provide mechanistic insight into how lipid A structural variation modulates macrophage immune programming and cytosolic inflammasome activation.