Diversity, Complexity, and Specificity of Bacterial Lipopolysaccharide (LPS) Structures Impacting Their Detection and Quantification.

Endotoxins are toxic lipopolysaccharides (LPSs), extending from the outer membrane of Gram-negative bacteria and notorious for their toxicity and deleterious effects. The comparison of different LPSs, isolated from various Gram-negative bacteria, shows a global similar architecture corresponding to a glycolipid lipid A moiety, a core oligosaccharide, and outermost long O-chain polysaccharides with molecular weights from 2 to 20 kDa. LPSs display high diversity and specificity among genera and species, and each bacterium contains a unique set of LPS structures, constituting its protective external barrier. Some LPSs are not toxic due to their particular structures. Different, well-characterized, and highly purified LPSs were used in this work to determine endotoxin detection rules and identify their impact on the host. Endotoxin detection is a major task to ensure the safety of human health, especially in the pharma and food sectors. Here, we describe the impact of different LPS structures obtained under different bacterial growth conditions on selective LPS detection methods such as LAL, HEK-blue TLR-4, LC-MS2, and MALDI-MS. In these various assays, LPSs were shown to respond differently, mainly attributable to their lipid A structures, their fatty acid numbers and chain lengths, the presence of phosphate groups, and their possible substitutions.

Manganese@Albumin Nanocomplex and Its Assembled Nanowire Activate TLR4-Dependent Signaling Cascades of Macrophages.

The immunomodulatory effect of divalent manganese cations (Mn2+ ), such as activation of the cGAS-STING pathway or NLRP3 inflammasomes, positions them as adjuvants for cancer immunotherapy. In this study, it is found that trace Mn2+ ions, bound to bovine serum albumin (BSA) to form Mn@BSA nanocomplexes, stimulate pro-inflammatory responses in human- or murine-derived macrophages through TLR4-mediated signaling cascades. Building on this, the assembly of Mn@BSA nanocomplexes to obtain nanowire structures enables stronger and longer-lasting immunostimulation of macrophages by regulating phagocytosis. Furthermore, Mn@BSA nanocomplexes and their nanowires efficiently activate peritoneal macrophages, reprogramme tumor-associated macrophages, and inhibit the growth of melanoma tumors in vivo. They also show better biosafety for potential clinical applications compared to typical TLR4 agonists such as lipopolysaccharides. Accordingly, the findings provide insights into the mechanism of metalloalbumin complexes as potential TLR agonists that activate macrophage polarization and highlight the importance of their nanostructures in regulating macrophage-mediated innate immunity.

Targeted Liposomal Co-delivery of an Immunogenic Cell Death Inducer and a Toll-Like Receptor 4 Agonist for Enhanced Cancer Chemo-immunotherapy.

Anticancer chemo-immunotherapy has gained considerable attention across various scientific domains as a prospective approach for the comprehensive eradication of malignant tumors. Recent research has particularly been focused on traditional anthracycline chemo drugs, such as doxorubicin and mitoxantrone. These compounds trigger apoptosis in tumor cells and evoke immunogenic cell death (ICD). ICD is a pivotal initiator of the cancer-immunity cycle by facilitating the release of damage-associated molecular patterns (DAMPs). The resultant DAMPs released from cancer cells effectively activate the immune system, resulting in an increase in tumor-infiltrating T cells. In this study, we have innovated a co-delivery strategy involving folate-modified liposomes to deliver doxorubicin and monophosphoryl lipid A (MPLA) simultaneously to tumor tissue. The engineered liposomes exploit the overexpression of folate receptors within the tumor tissues. Delivered doxorubicin initiates ICD at the tumor cells, further enhancing the immunogenic stimulus. Additionally, MPLA helps T cell priming by activating antigen-presenting cells. This intricate interplay culminates in a synergistic effect, ultimately resulting in an augmented and potentiated anticancer chemo-immunotherapeutic liposomal treatment.

CRX-527 induced differentiation of HSCs protecting the intestinal epithelium from radiation damage.

Recently, Toll-like receptors (TLRs) have been extensively studied in radiation damage, but the inherent defects of high toxicity and low efficacy of most TLR ligands limit their further clinical transformation. CRX-527, as a TLR4 ligand, has rarely been reported to protect against radiation. We demonstrated that CRX-527 was safer than LPS at the same dose in vivo and had almost no toxic effect in vitro. Administration of CRX-527 improved the survival rate of total body irradiation (TBI) to 100% in wild-type mice but not in TLR4-/- mice. After TBI, hematopoietic system damage was significantly alleviated, and the recovery period was accelerated in CRX-527-treated mice. Moreover, CRX-527 induced differentiation of HSCs and the stimulation of CRX-527 significantly increased the proportion and number of LSK cells and promoted their differentiation into macrophages, activating immune defense. Furthermore, we proposed an immune defense role for hematopoietic differentiation in the protection against intestinal radiation damage, and confirmed that macrophages invaded the intestines through peripheral blood to protect them from radiation damage. Meanwhile, CRX-527 maintained intestinal function and homeostasis, promoted the regeneration of intestinal stem cells, and protected intestinal injury from lethal dose irradiation. Furthermore, After the use of mice, we found that CRX-527 had no significant protective effect on the hematopoietic and intestinal systems of irradiated TLR4-/- mice. in conclusion, CRX-527 induced differentiation of HSCs protecting the intestinal epithelium from radiation damage.

Immunomodulatory Properties of a γ-Aminobutyric Acid-Enriched Strawberry Juice Produced by Levilactobacillus brevis CRL 2013.

Gamma-aminobutyric acid (GABA) plays a key role in mammals as the major inhibitory neurotransmitter of the central nervous system. Although GABA may not be able to cross the human blood-brain barrier, it was approved as a food ingredient because of its benefits to the host after oral administration including anti-hypertensive, anti-depressant and anti-inflammatory activities. Considering the current trend toward the development of new functional and natural products and that microbial fermentation is one of the most promising methods to produce this non-protein amino acid, the in situ production of GABA through fermentation of strawberry and blueberry juices by the efficient GABA producer strain, Levilactobacillus brevis (formerly known as Lactobacillus brevis) CRL 2013, was evaluated. A high GABA production (262 mM GABA) was obtained after fermenting strawberry juice supplemented with yeast extract for 168 h, being GABA yield significantly higher in strawberry juices than in the blueberry ones. Thus, GABA-enriched fermented strawberry juice (FSJ) was selected to carry out in vivo and in vitro studies. The in vitro functional analysis of the GABA-enriched FSJ demonstrated its ability to significantly decrease the expression of cox-2 gene in LPS stimulated RAW 264.7 macrophages. In addition, in vivo studies in mice demonstrated that both, L. brevis CRL 2013 and the GABA-enriched FSJ were capable of reducing the levels of peritoneal, intestinal and serum TNF-α, IL-6, and CXCL1, and increasing IL-10 and IFN-γ in mice exposed to an intraperitoneal challenge of LPS. Of note, the GABA-enriched FSJ was more efficient than the CRL 2013 strain to reduce the pro-inflammatory factors and enhance IL-10 production. These results indicated that the CRL 2013 strain exerts anti-inflammatory effects in the context of LPS stimulation and that this effect is potentiated by fermentation. Our results support the potential use of L. brevis CRL 2013 as an immunomodulatory starter culture and strawberry juice as a remarkable vegetable matrix for the manufacture of GABA-enriched fermented functional foods capable of differentially modulating the inflammatory response triggered by TLR4 activation.