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Supplemental material, sj-pdf-1-ini-10.1177_17534259211005840 for Synthetic glycolipid-based TLR4 antagonists negatively regulate TRIF-dependent TLR4 signalling in human macrophages by Charys Palmer, Fabio A Facchini, Richard PO Jones, Frank Neumann, Francesco Peri and Grisha Pirianov in Innate Immunity

I recettori Toll-like (TLR) sono attivati in molte interazioni ospite-patogeno. Essi riconoscono pattern molecolari associati a patogeni (PAMP) inducendo la risposta immunitaria innata nell’ospite. Il TLR4 permette ai mammiferi di rilevare l’endotossina batterica, lipopolisaccaride (LPS). L’attivazione incontrollata del TLR4 è alla base di sepsi sistemica acuta e di molti disturbi infiammatori, come le malattie infiammatorie intestinali (IBD). La modulazione del TLR4 è pertanto di estremo interesse terapeutico. Questa tesi di dottorato si basa su tre articoli (capitolo 1-3) e ha lo scopo di studiare il potenziale ruolo terapeutico di piccole molecole attive sul TLR4, testate da sole o in combinazione con peptidi antimicrobici (AMP). Nel capitolo I mostriamo la progettazione e la caratterizzazione biologica di una serie di molecole anfifiliche con scaffold calixarenico. La struttura di questi composti è stata progettata mediante studi computazionali in modo da poter interagire con il complesso TLR4/MD-2 e con il co-recettore CD14. Alcuni calixareni ottenuti inibiscono l'attivazione di TLR4 indotta da LPS in modo dose-dipendente e riducono la produzione di citochine mediata da TLR4 in cellule immunitarie umane e murine. Inoltre, i guanidinocalixareni cationici sono in grado di inibire l’attivazione del TLR4 indotta dalla lectina PHA. Sebbene l’attività inibitoria dei guanidinocalixareni è stata correlata alla capacità di neutralizzare l’LPS, i risultati ottenuti in questo studio suggeriscono un effetto tali molecole diretto sul complesso TLR4/MD-2. La struttura dei calixareni potrebbe pertanto rivelarsi utile per lo sviluppo di nuovi modulatori del TLR4. Nel capitolo II sono riportati gli effetti della co-somministrazione di peptidi antimicrobici (AMP) con un antagonista sintetico del TLR4 (FP7) sull'attivazione del TLR4. Due AMP noti per neutralizzare LPS aumentano di un ordine di grandezza la potenza di FP7 nel bloccare il segnale mediato da TLR4. Questo effetto è mantenuto anche quando le cellule sono stimolate con un agonista del TLR4 diverso da LPS. I dati suggeriscono un possibile doppio meccanismo d’azione, non esclusivamente basato sulla capacità degli AMP di neutralizzare l’LPS, ma anche ad un effetto diretto sui recettori dell’endotossina. Esperimenti NMR in soluzione mostrano che l'aggiunta degli AMP modifica lo stato di aggregazione di FP7 promuovendo la formazione di micelle più grandi. Questi risultati suggeriscono una relazione tra lo stato di aggregazione di ligandi mimetici dell’LPS e il tipo/intensità della risposta mediata da TLR4. Il capitolo III descrive uno studio preclinico in cui l’antagonista FP7 è stato utilizzato in un modello sperimentale di malattia infiammatoria intestinale (IBD). Questo studio ha lo scopo di valutare una possibile strategia terapeutica basata sull'uso di piccole molecole dirette selettivamente sul complesso TLR4/MD-2 per ridurre l'infiammazione nelle IBD. I risultati ottenuti mostrano che FP7 riduce la secrezione di citochine proinfiammatorie indotta da LPS da parte di cellule mononucleate del sangue periferico (PBMC) e di cellule mononucleate della lamina propria (LPMC) isolate dai pazienti con IBD. L'effetto anti-infiammatorio di FP7 è dovuto ad una ridotta attivazione della via di segnalazione indotta da LPS dipendente dal fattore myeloid differentiation primary response gene (88) (Myd88). Lo studio mostra che il meccanismo di azione di FP7 è correlato alla sua capacità di competere con l’LPS per il legame con i recettori MD-2 e CD14. Inoltre FP7 riduce l'infiammazione in vivo su un modello murino di colite ulcerosa. Considerando che la patogenesi delle IBD è associata a un’eccessiva risposta immunitaria innata verso antigeni microbici, l'inibizione del TLR4 da parte di agenti chimici come FP7 potrebbe rivelarsi un promettente approccio alternativo al trattamento delle IBD. Among the first receptors activated during host-pathogen interactions are Toll-like receptors (TLRs), which detect pathogen-associated molecular patterns (PAMPs) to induce innate and adaptive immune responses. TLR4 is the mammalian sensor of bacterial endotoxin, lipopolysaccharide (LPS). Dysregulated TLR4 activation is involved in acute systemic sepsis and in many disorders that involve inflammation, including inflammatory bowel diseases (IBDs). Therefore, therapeutic modulation of TLR4 signalling is of major interest. This PhD thesis is based on three papers (Chapter 1-3) and has the aim to study the capacity of synthetic small molecule TLR4 antagonists, alone or in combination with antimicrobial peptides (AMPs), to act as therapeutics in inflammatory diseases. In Chapter I, starting from the assumption that opportunely designed cationic amphiphiles can behave as ligands of the LPS receptor system and therefore modulate the TLR4 signaling, we present the rational design and biological characterization of a panel of amphiphilic guanidinocalixarenes. The structure of these compounds was computationally designed and optimized to dock into MD-2 and CD14 binding sites. We found that some of these calixarenes were active in inhibiting, in a dose-dependent way, the LPS-stimulated TLR4 activation and TLR4-dependent cytokine production in human and mouse immune cells. Moreover, cationic guanidinocalixarenes also inhibited TLR4 signaling when TLR4 was activated by the plant lectin PHA. While the activity of guanidinocalixarenes in inhibiting LPS toxic action has previously been related to their capacity to bind and neutralize LPS, the results obtained in this chapter suggest a direct antagonist effect of calixarenes on TLR4/MD-2 dimerization: this suggests the use of the calixarene scaffold for the development of new TLR4-directed therapeutics. In Chapter II is presented the effect of co-administration of antimicrobial peptides (AMPs) and a synthetic TLR4 antagonist (the glycolipid FP7) on TLR4 activation and signalling. The co-administration of two LPS-neutralizing peptides (a cecropin A-melittin hybrid peptide and a human cathelicidin) enhances by an order of magnitude the potency of FP7 in blocking the TLR4 signal. Interestingly, this potentiation effect also occurs when cells are stimulated with a non-LPS TLR4 agonist. Our data suggest a dual mechanism of action for the peptide/glycolipid combination, not exclusively based on LPS binding and neutralization, but also on a direct effect on CD14 and MD-2 binding. NMR experiments in solution show that peptide addition changes the aggregation state of FP7, promoting the formation of larger micelles. These results suggest a relationship between the aggregation state of lipid A-like ligands and the type and intensity of TLR4 response. Chapter III describes a preclinical study in which FP7 is used in an experimental model of IBD. This study has the aim to evaluate a possible therapeutic strategy based on the use of small molecule that selectively targets TLR4/MD-2 complex to reduce IBD inflammation. The results obtained show that FP7 reduced the secretion of the main LPS-induced innate pro-inflammatory cytokines by peripheral blood mononuclear cells (PBMCs) and lamina propria mononuclear cells (LPMCs) isolated from IBD patients. FP7 anti-inflammatory effect is due to a reduced activation of the main myeloid differentiation primary response gene (88) (Myd88)-depedent pathway effectors normally induced by LPS presence. We indicated that the mechanism of action of FP7 is related to its capacity to compete with LPS for the binding to MD-2 adaptor protein and to CD14 co-receptor. FP7 also reduced inflammation in vivo on a murine model of ulcerative colitis. Considering that IBD pathogenesis is associated to an abnormal innate immune response towards microbial antigens, TLR4 inhibition by chemical agents as FP7 emerged as a promising alternative approach to IBD treatment.

The structure-activity relationship was investigated in a series of synthetic TLR4 antagonists formed by a glucosamine core linked to two phosphate esters and two linear carbon chains. Molecular modeling showed that the compounds with 10, 12 and 14 carbons chains are associated to higher stabilization of the MD-2/TLR4 antagonist conformation than in the case of the C16 variant. Binding experiments with human MD-2 showed that the C12 and C14 variants have higher affinity than C10, while the C16 variant did not interact with the protein.\ud The molecules, with the exception of the C16 variant, inhibited the LPS-stimulated TLR4 signal in human and murine cells and the antagonist potency mirrored the MD-2 affinity calculated from in vitro binding experiments. FT-IR, NMR, and SAXS measurements suggested that the aggregation state in aqueous solution depends on fatty acid chains lengths and that this property can influence TLR4 activity in this series of compounds.

Journal of medicinal chemistry 61(7), 2895 - 2909 (2018). doi:10.1021/acs.jmedchem.7b01803, The structure–activity relationship was investigated in a series of synthetic TLR4 antagonists formed by a glucosamine core linked to two phosphate esters and two linear carbon chains. Molecular modeling showed that the compounds with 10, 12, and 14 carbons chains are associated with higher stabilization of the MD-2/TLR4 antagonist conformation than in the case of the C16 variant. Binding experiments with human MD-2 showed that the C12 and C14 variants have higher affinity than C10, while the C16 variant did not interact with the protein. The molecules, with the exception of the C16 variant, inhibited the LPS-stimulated TLR4 signal in human and murine cells, and the antagonist potency mirrored the MD-2 affinity calculated from in vitro binding experiments. Fourier-transform infrared, nuclear magnetic resonance, and small angle X-ray scattering measurements suggested that the aggregation state in aqueous solution depends on fatty acid chain lengths and that this property can influence TLR4 activity in this series of compounds., Published by ACS, Washington, DC

12 p.-7 fig.-1 schem.-1 graph. abst., Modern adjuvants for vaccine formulations are immunostimulating agents whose action is based on the activation of pattern recognition receptors (PRRs) by well-defined ligands to boost innate and adaptive immune responses. Monophosphoryl lipid A (MPLA), a detoxified analogue of lipid A, is a clinically approved adjuvant that stimulates toll-like receptor 4 (TLR4). The synthesis of MPLA poses manufacturing and quality assessment challenges. Bridging this gap, we report here the development and preclinical testing of chemically simplified TLR4 agonists that could sustainably be produced in high purity and on a large scale. Underpinned by computational and biological experiments, we show that synthetic monosaccharide-based molecules (FP compounds) bind to the TLR4/MD-2 dimer with submicromolar affinities stabilizing the active receptor conformation. This results in the activation of MyD88- and TRIF-dependent TLR4 signaling and the NLRP3 inflammasome. FP compounds lack in vivo toxicity and exhibit adjuvant activity by stimulating antibody responses with a potency comparable to MPLA., The support from European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 860325 (www.BactiVax.eu), Italian Ministry grant PRIN 2017, no. 2017E44A9P (BacHounds:Supramolecular nanostructures for bacteria detection), and the Spanish Ministry for Science and Innovation (grants CTQ2017-88353-R and PRE2018-086249) is acknowledged.

Severe COVID-19 is characterized by overproduction of immune mediators, but the role of interferons (IFNs) of the type I (IFN-I) or type III (IFN-III) families remains debated. We scrutinized the production of IFNs along the respiratory tract of COVID-19 patients and found that high levels of IFN-III, and to a lesser extent IFN-I, characterize the upper airways of patients with high viral burden but reduced disease risk or severity. Production of specific IFN-III, but not IFN-I, members, denotes patients with a mild pathology and efficiently drives the transcription of genes that protect against SARS-CoV-2. In contrast, compared to subjects with other infectious or non-infectious lung pathologies, IFNs are over-represented in the lower airways of patients with severe COVID-19 that exhibit gene pathways associated with increased apoptosis and decreased proliferation. Our data demonstrate a dynamic production of IFNs in SARS-CoV-2-infected patients and show IFNs play opposing roles at distinct anatomical sites., An in-depth analysis of interferons in COVID-19 reveals differences in their roles based on anatomical location, viral load, age and disease severity. In the upper respiratory tract, high levels of IFN-III are protective and result in mild disease in spite of higher SARS-CoV-2 viral burden while the lower airways of patients with severe COVID-19 demonstrate elevated IFN-I and III, cell death and a reduction in interferon stimulated genes.

Growing evidence suggests that conventional dendritic cells (cDCs) undergo aberrant maturation in COVID‐19, which negatively affects T‐cell activation. The presence of effector T cells in patients with mild disease and dysfunctional T cells in severely ill patients suggests that adequate T‐cell responses limit disease severity. Understanding how cDCs cope with SARS‐CoV‐2 can help elucidate how protective immune responses are generated. Here, we report that cDC2 subtypes exhibit similar infection‐induced gene signatures, with the upregulation of interferon‐stimulated genes and interleukin (IL)‐6 signaling pathways. Furthermore, comparison of cDCs between patients with severe and mild disease showed severely ill patients to exhibit profound downregulation of genes encoding molecules involved in antigen presentation, such as MHCII, TAP, and costimulatory proteins, whereas we observed the opposite for proinflammatory molecules, such as complement and coagulation factors. Thus, as disease severity increases, cDC2s exhibit enhanced inflammatory properties and lose antigen presentation capacity. Moreover, DC3s showed upregulation of anti‐apoptotic genes and accumulated during infection. Direct exposure of cDC2s to the virus in vitro recapitulated the activation profile observed in vivo. Our findings suggest that SARS‐CoV‐2 interacts directly with cDC2s and implements an efficient immune escape mechanism that correlates with disease severity by downregulating crucial molecules required for T‐cell activation. This article is protected by copyright. All rights reserved

Acetobacter pasteurianus is an acetic acid-producing Gram-negative bacterium commonly found associated with plants and plant products and widely used in the production of fermented foods, such as kefir and vinegar. Due to the acid conditions of the bacterium living habitat, uncommon structural features composing its cell envelope are expected. In the present work we have investigated the A. pasteurianus CIP103108 lipopolysaccharide (LPS) structure and immunoactivity. The structure of the lipid A and of two different O-polysaccharides was assessed. Furthermore, immunological studies with human cells showed a low immunostimulant activity of the isolated LPS, in addition to a slight capability to lower the NF-kB activation upon stimulation by toxic LPS.

The COVID-19 outbreak driven by SARS-CoV-2 has caused more than 2.5 million deaths globally, with the most severe cases characterized by over-exuberant production of immune-mediators, the nature of which is not fully understood. Interferons of the type I (IFN-I) or type III (IFN-III) families are potent antivirals, but their role in COVID-19 remains debated. Our analysis of gene and protein expression along the respiratory tract shows that IFNs, especially IFN-III, are over-represented in the lower airways of patients with severe COVID-19, while high levels of IFN-III, and to a lesser extent IFN-I, characterize the upper airways of patients with high viral burden but reduced disease risk or severity; also, IFN expression varies with abundance of the cell types that produce them. Our data point to a dynamic process of inter- and intra-family production of IFNs in COVID-19, and suggest that IFNs play opposing roles at distinct anatomical sites.