An antibody to IL-1 receptor 7 protects mice from LPS-induced tissue and systemic inflammation.

Introduction: Interleukin-18 (IL-18), a pro-inflammatory cytokine belonging to the IL-1 Family, is a key mediator ofautoinflammatory diseases associated with the development of macrophage activation syndrome (MAS).High levels of IL-18 correlate with MAS and COVID-19 severity and mortality, particularly in COVID-19patients with MAS. As an inflammation inducer, IL-18 binds its receptor IL-1 Receptor 5 (IL-1R5), leadingto the recruitment of the co-receptor, IL-1 Receptor 7 (IL-1R7). This heterotrimeric complex subsequentlyinitiates downstream signaling, resulting in local and systemic inflammation. Methods: We reported earlier the development of a novel humanized monoclonal anti-human IL-1R7 antibody whichspecifically blocks the activity of human IL-18 and its inflammatory signaling in human cell and wholeblood cultures. In the current study, we further explored the strategy of blocking IL-1R7 inhyperinflammation in vivo using animal models. Results: We first identified an anti-mouse IL-1R7 antibody that significantly suppressed mouse IL-18 andlipopolysaccharide (LPS)-induced IFNg production in mouse splenocyte and peritoneal cell cultures. Whenapplied in vivo, the antibody reduced Propionibacterium acnes and LPS-induced liver injury and protectedmice from tissue and systemic hyperinflammation. Importantly, anti-IL-1R7 significantly inhibited plasma,liver cell and spleen cell IFNg production. Also, anti-IL-1R7 downregulated plasma TNFa, IL-6, IL-1b,MIP-2 production and the production of the liver enzyme ALT. In parallel, anti-IL-1R7 suppressed LPSinducedinflammatory cell infiltration in lungs and inhibited the subsequent IFNg production andinflammation in mice when assessed using an acute lung injury model. Discussion: Altogether, our data suggest that blocking IL-1R7 represents a potential therapeutic strategy to specificallymodulate IL-18-mediated hyperinflammation, warranting further investigation of its clinical application intreating IL-18-mediated diseases, including MAS and COVID-19.

Metabolic reprogramming of the inflammatory response in the nervous system: the crossover between inflammation and metabolism.

Metabolism is a fundamental process by which biochemicals are broken down to produce energy (catabolism) or used to build macromolecules (anabolism). Metabolism has received renewed attention as a mechanism that generates molecules that modulate multiple cellular responses. This was first identified in cancer cells as the Warburg effect, but it is also present in immunocompetent cells. Studies have revealed a bidirectional influence of cellular metabolism and immune cell function, highlighting the significance of metabolic reprogramming in immune cell activation and effector functions. Metabolic processes such as glycolysis, oxidative phosphorylation, and fatty acid oxidation have been shown to undergo dynamic changes during immune cell response, facilitating the energetic and biosynthetic demands. This review aims to provide a better understanding of the metabolic reprogramming that occurs in different immune cells upon activation, with a special focus on central nervous system disorders. Understanding the metabolic changes of the immune response not only provides insights into the fundamental mechanisms that regulate immune cell function but also opens new approaches for therapeutic strategies aimed at manipulating the immune system.

IL-38 regulates intestinal stem cell homeostasis by inducing WNT signaling and beneficial IL-1ß secretion.

The IL-1 Family member IL-38 has been characterized primarily as an antiinflammatory cytokine in human and mouse models of systemic diseases. Here, we examined the role of IL-38 in the murine small intestine (SI). Immunostaining of SI revealed that IL-38 expression partially confines to intestinal stem cells. Cultures of intestinal organoids reveal IL-38 functions as a growth factor by increasing organoid size via inducing WNT3a. In contrast, organoids from IL-38-deficient mice develop more slowly. This reduction in size is likely due to the downregulation of intestinal stemness markers (i.e., Fzd5, Ephb2, and Olfm4) expression compared with wild-type organoids. The IL-38 binding to IL-1R6 and IL-1R9 is still a matter of debate. Therefore, to analyze the molecular mechanisms of IL-38 signaling, we also examined organoids from IL-1R9-deficient mice. Unexpectedly, these organoids, although significantly smaller than wild type, respond to IL-38, suggesting that IL-1R9 is not involved in IL-38 signaling in the stem cell crypt. Nevertheless, silencing of IL-1R6 disabled the organoid response to the growth property of IL-38, thus suggesting IL-1R6 as the main receptor used by IL-38 in the crypt compartment. In organoids from wild-type mice, IL-38 stimulation induced low concentrations of IL-1ß which contribute to organoid growth. However, high concentrations of IL-1ß have detrimental effects on the cultures that were prevented by treatment with recombinant IL-38. Overall, our data demonstrate an important regulatory function of IL-38 as a growth factor, and as an antiinflammatory molecule in the SI, maintaining homeostasis.

Pancreatic Ductal Adenocarcinoma Cells Regulate NLRP3 Activation to Generate a Tolerogenic Microenvironment.

Defining feature of pancreatic ductal adenocarcinoma (PDAC) that participates in the high mortality rate and drug resistance is the immune-tolerant microenvironment which enables tumors to progress unabated by adaptive immunity. In this study, we report that PDAC cells release CSF-1 to induce nucleotide-binding domain, leucine-rich containing family, pyrin domain-containing-3 (NLRP3) activation in myeloid cells. Increased NLRP3 expression was found in the pancreas of patients with PDAC when compared with normal pancreas which correlated with the formation of the NLRP3 inflammasome. Using human primary cells and an orthotopic PDAC mouse model, we show that NLRP3 activation is responsible for the maturation and release of the inflammatory cytokine IL1ß which selectively drives Th2-type inflammation via COX2/PGE2 induction. As a result of this inflammation, primary tumors were characterized by reduced cytotoxic CD8+ T-cell activation and increased tumor expansion. Genetic deletion and pharmacologic inhibition of NLRP3 enabled the development of Th1 immunity, increased intratumoral levels of IL2, CD8+ T cell­mediated tumor suppression, and ultimately limited tumor growth. In addition, we observed that NLRP3 inhibition in combination with gemcitabine significantly increased the efficacy of the chemotherapy. In conclusion, this study provides a mechanism by which tumor-mediated NLRP3 activation exploits a distinct adaptive immunity response that facilitates tumor escape and progression. Considering the ability to block NLRP3 activity with safe and small orally active molecules, this protein represents a new promising target to improve the limited therapeutic options in PDAC. SIGNIFICANT: This study provides novel molecular insights on how PDAC cells exploit NLRP3 activation to suppress CD8 T-cell activation. From a translational perspective, we demonstrate that the combination of gemcitabine with the orally active NLRP3 inhibitor OLT1177 increases the efficacy of monotherapy.

Pharmacologic inhibition of NLRP3 reduces the levels of α-synuclein and protects dopaminergic neurons in a model of Parkinson's disease.

BACKGROUND: Parkinson's disease (PD) is characterized by a progressive degeneration of dopaminergic neurons, which leads to irreversible loss of peripheral motor functions. Death of dopaminergic neurons induces an inflammatory response in microglial cells, which further exacerbates neuronal loss. Reducing inflammation is expected to ameliorate neuronal loss and arrest motor dysfunctions. Because of the contribution of the NLRP3 inflammasome to the inflammatory response in PD, we targeted NLRP3 using the specific inhibitor OLT1177®. METHODS: We evaluated the effectiveness of OLT1177® in reducing the inflammatory response in an MPTP neurotoxic model of PD. Using a combination of in vitro and in vivo studies, we analyzed the effects of NLRP3 inhibition on pro-inflammatory markers in the brain, α-synuclein aggregation, and dopaminergic neuron survival. We also determined the effects of OLT1177® on locomotor deficits associated with MPTP and brain penetrance. RESULTS: Treatment with OLT1177® prevented the loss of motor function, reduced the levels of α-synuclein, modulated pro-inflammatory markers in the nigrostriatal areas of the brain, and protected dopaminergic neurons from degeneration in the MPTP model of PD. We also demonstrated that OLT1177® crosses the blood-brain barrier and reaches therapeutic concentrations in the brain. CONCLUSIONS: These data suggest that targeting the NLRP3 inflammasome by OLT1177® may be a safe and novel therapeutic approach to arrest neuroinflammation and protect against neurological deficits of Parkinson's disease in humans.

Modulation of Multiple Gene Clusters' Expression by the PAS-LuxR Transcriptional Regulator PteF.

PAS-LuxR transcriptional regulators are conserved proteins governing polyene antifungal biosynthesis. PteF is the regulator of filipin biosynthesis from Streptomyces avermitilis. Its mutation drastically abates filipin, but also oligomycin production, a macrolide ATP-synthase inhibitor, and delays sporulation; thus, it has been considered a transcriptional activator. Transcriptomic analyses were performed in S. avermitilis DpteF and its parental strain. Both strains were grown in a YEME medium without sucrose, and the samples were taken at exponential and stationary growth phases. A total of 257 genes showed an altered expression in the mutant, most of them at the exponential growth phase. Surprisingly, despite PteF being considered an activator, most of the genes affected showed overexpression, thereby suggesting a negative modulation. The affected genes were related to various metabolic processes, including genetic information processing; DNA, energy, carbohydrate, and lipid metabolism; morphological differentiation; and transcriptional regulation, among others, but were particularly related to secondary metabolite biosynthesis. Notably, 10 secondary metabolite gene clusters out of the 38 encoded by the genome showed altered expression profiles in the mutant, suggesting a regulatory role for PteF that is wider than expected. The transcriptomic results were validated by quantitative reverse-transcription polymerase chain reaction. These findings provide important clues to understanding the intertwined regulatory machinery that modulates antibiotic biosynthesis in Streptomyces.

IL-38 Gene Deletion Worsens Murine Colitis.

IL-38 is a recently discovered cytokine and member of the IL-1 Family. In the IL-1 Family, IL-38 is unique because the cytokine is primarily a B lymphocyte product and functions to suppress inflammation. Studies in humans with inflammatory bowel disease (IBD) suggest that IL-38 may be protective for ulcerative colitis or Crohn's disease, and that IL-38 acts to maintain homeostasis in the intestinal tract. Here we investigated the role of endogenous IL-38 in experimental colitis in mice deficient in IL-38 by deletion of exons 1-4 in C57 BL/6 mice. Compared to WT mice, IL-38 deficient mice subjected to dextran sulfate sodium (DSS) showed greater severity of disease, more weight loss, increased intestinal permeability, and a worse histological phenotype including increased neutrophil influx in the colon. Mice lacking IL-38 exhibited elevated colonic Nlrp3 mRNA and protein levels, increased caspase-1 activation, and the concomitant increased processing of IL-1ß precursor into active IL-1ß. Expression of IL-1α, an exacerbator of IBD, was also upregulated. Colonic myleloperoxidase protein and Il17a, and Il17f mRNA levels were higher in the IL-38 deficient mice. Daily treatment of IL-38 deficient mice with an NLRP3 inhibitor attenuated diarrhea and weight loss during the recovery phase. These data implicate endogenous IL-38 as an anti-inflammatory cytokine that reduces DSS colitis severity. We propose that a relative deficiency of IL-38 contributes to IBD by disinhibition of the NLRP3 inflammasome.

CD200R1 Contributes to Successful Functional Reinnervation after a Sciatic Nerve Injury.

Activating and inhibitory immune receptors play a critical role in regulating systemic and central nervous system (CNS) immune and inflammatory processes. The CD200R1 immunoreceptor induces a restraining signal modulating inflammation and phagocytosis in the CNS under different inflammatory conditions. However, it remains unknown whether CD200R1 has a role in modulating the inflammatory response after a peripheral nerve injury, an essential component of the successful regeneration. Expression of CD200R1 and its ligand CD200 was analyzed during homeostasis and after a sciatic nerve crush injury in C57Bl/6 mice. The role of CD200R1 in Wallerian Degeneration (WD) and nerve regeneration was studied using a specific antibody against CD200R1 injected into the nerve at the time of injury. We found an upregulation of CD200R1 mRNA after injury whereas CD200 was downregulated acutely after nerve injury. Blockade of CD200R1 significantly reduced the acute entrance of both neutrophils and monocytes from blood after nerve injury. When long term regeneration and functional recovery were evaluated, we found that blockade of CD200R1 had a significant effect impairing the spontaneous functional recovery. Taken together, these results show that CD200R1 has a role in mounting a successful acute inflammatory reaction after injury, and contributes to an effective functional recovery.

Understanding microRNAs in the Context of Infection to Find New Treatments against Human Bacterial Pathogens.

The development of RNA-based anti-infectives has gained interest with the successful application of mRNA-based vaccines. Small RNAs are molecules of RNA of <200 nucleotides in length that may control the expression of specific genes. Small RNAs include small interference RNAs (siRNAs), Piwi-interacting RNAs (piRNAs), or microRNAs (miRNAs). Notably, the role of miRNAs on the post-transcriptional regulation of gene expression has been studied in detail in the context of cancer and many other genetic diseases. However, it is also becoming apparent that some human miRNAs possess important antimicrobial roles by silencing host genes essential for the progress of bacterial or viral infections. Therefore, their potential use as novel antimicrobial therapies has gained interest during the last decade. The challenges of the transport and delivery of miRNAs to target cells are important, but recent research with exosomes is overcoming the limitations in RNA-cellular uptake, avoiding their degradation. Therefore, in this review, we have summarised the latest developments in the exosomal delivery of miRNA-based therapies, which may soon be another complementary treatment to pathogen-targeted antibiotics that could help solve the problem caused by multidrug-resistant bacteria.

Evaluation of Natural Language Processing for the Identification of Crohn Disease-Related Variables in Spanish Electronic Health Records: A Validation Study for the PREMONITION-CD Project.

BACKGROUND: The exploration of clinically relevant information in the free text of electronic health records (EHRs) holds the potential to positively impact clinical practice as well as knowledge regarding Crohn disease (CD), an inflammatory bowel disease that may affect any segment of the gastrointestinal tract. The EHRead technology, a clinical natural language processing (cNLP) system, was designed to detect and extract clinical information from narratives in the clinical notes contained in EHRs. OBJECTIVE: The aim of this study is to validate the performance of the EHRead technology in identifying information of patients with CD. METHODS: We used the EHRead technology to explore and extract CD-related clinical information from EHRs. To validate this tool, we compared the output of the EHRead technology with a manually curated gold standard to assess the quality of our cNLP system in detecting records containing any reference to CD and its related variables. RESULTS: The validation metrics for the main variable (CD) were a precision of 0.88, a recall of 0.98, and an F1 score of 0.93. Regarding the secondary variables, we obtained a precision of 0.91, a recall of 0.71, and an F1 score of 0.80 for CD flare, while for the variable vedolizumab (treatment), a precision, recall, and F1 score of 0.86, 0.94, and 0.90 were obtained, respectively. CONCLUSIONS: This evaluation demonstrates the ability of the EHRead technology to identify patients with CD and their related variables from the free text of EHRs. To the best of our knowledge, this study is the first to use a cNLP system for the identification of CD in EHRs written in Spanish.

Inhibition of the NLRP3 inflammasome by OLT1177 induces functional protection and myelin preservation after spinal cord injury.

Spinal cord injury (SCI) leads to irreversible functional deficits due to the disruption of axons and the death of neurons and glial cells. The inflammatory response that occurs in the injured spinal cord results in tissue degeneration; thus, targeting inflammation after acute SCI is expected to ameliorate histopathological evidence indicative of damage and, consequently, reduce functional disabilities. Interleukin 1 beta (IL-1ß) and interleukin 18 (IL-18) are pro-inflammatory cytokines members of the IL-1 family that initiate and propagate inflammation. Here, we report that protein levels of IL-1ß and IL-18 were increased in spinal cord parenchyma after SCI, but with different expression profiles. Whereas levels of IL-1ß were rapidly increased reaching peak levels at 12 h after the injury, levels of IL-18 did not increase until 7 days after the injury. Since activation of the NLRP3 inflammasome is required for the processing and release of IL-1ß and IL-18, we intraperitoneally administered OLT1177, a selective inhibitor of the NLRP3 inflammasome, to reduce the contribution of these cytokines to SCI. At a dose of 200 mg/kg, OLT1177 protected against neurological deficits and histological evidence of damage. OLT1177 also reduced the levels of IL-1ß in the spinal cord after contusion injury and diminished the accumulation of neutrophils and macrophages at later time points. These data suggest that targeting the NLRP3 inflammasome with OLT1177 could be a novel therapeutic strategy to arrest neuroinflammation and reduce functional impairments after acute SCI in humans.

Clinical characteristics and prognostic factors for Crohn's disease relapses using natural language processing and machine learning: a pilot study.

BACKGROUND: The impact of relapses on disease burden in Crohn's disease (CD) warrants searching for predictive factors to anticipate relapses. This requires analysis of large datasets, including elusive free-text annotations from electronic health records. This study aims to describe clinical characteristics and treatment with biologics of CD patients and generate a data-driven predictive model for relapse using natural language processing (NLP) and machine learning (ML). METHODS: We performed a multicenter, retrospective study using a previously validated corpus of CD patient data from eight hospitals of the Spanish National Healthcare Network from 1 January 2014 to 31 December 2018 using NLP. Predictive models were created with ML algorithms, namely, logistic regression, decision trees, and random forests. RESULTS: CD phenotype, analyzed in 5938 CD patients, was predominantly inflammatory, and tobacco smoking appeared as a risk factor, confirming previous clinical studies. We also documented treatments, treatment switches, and time to discontinuation in biologics-treated CD patients. We found correlations between CD and patient family history of gastrointestinal neoplasms. Our predictive model ranked 25 000 variables for their potential as risk factors for CD relapse. Of highest relative importance were past relapses and patients' age, as well as leukocyte, hemoglobin, and fibrinogen levels. CONCLUSION: Through NLP, we identified variables such as smoking as a risk factor and described treatment patterns with biologics in CD patients. CD relapse prediction highlighted the importance of patients' age and some biochemistry values, though it proved highly challenging and merits the assessment of risk factors for relapse in a clinical setting.

Interleukin-4 and interleukin-13 induce different metabolic profiles in microglia and macrophages that relate with divergent outcomes after spinal cord injury.

Background: Microglia and macrophages adopt a pro-inflammatory phenotype after spinal cord injury (SCI), what is thought to contribute to secondary tissue degeneration. We previously reported that this is due, in part, to the low levels of anti-inflammatory cytokines, such as IL-4. Since IL-13 and IL-4 share receptors and both cytokines drive microglia and macrophages towards an anti-inflammatory phenotype in vitro, here we studied whether administration of IL-13 and IL-4 after SCI leads to beneficial effects. Methods: We injected mice with recombinant IL-13 or IL-4 at 48 h after SCI and assessed their effects on microglia and macrophage phenotype and functional outcomes. We also performed RNA sequencing analysis of macrophages and microglia sorted from the injured spinal cords of mice treated with IL-13 or IL-4 and evaluated the metabolic state of these cells by using Seahorse technology. Results: We observed that IL-13 induced the expression of anti-inflammatory markers in microglia and macrophages after SCI but, in contrast to IL-4, it failed to mediate functional recovery. We found that these two cytokines induced different gene signatures in microglia and macrophages after SCI and that IL-4, in contrast to IL-13, shifted microglia and macrophage metabolism from glycolytic to oxidative phosphorylation. These findings were further confirmed by measuring the metabolic profile of these cells. Importantly, we also revealed that macrophages stimulated with IL-4 or IL-13 are not deleterious to neurons, but they become cytotoxic when oxidative metabolism is blocked. This suggests that the metabolic shift, from glycolysis to oxidative phosphorylation, is required to minimize the cytotoxic responses of microglia and macrophages. Conclusions: These results reveal that the metabolic fitness of microglia and macrophages after SCI contributes to secondary damage and that strategies aimed at boosting oxidative phosphorylation might be a novel approach to minimize the deleterious actions of microglia and macrophages in neurotrauma.

Production, Detection, Extraction, and Quantification of Polyene Antibiotics.

Polyene antibiotics are macrolide antifungal compounds obtained by fermentation of producer Streptomyces strains. Here we describe commonly used methods for polyene production, detection, and their subsequent extraction and purification. While bioassays are used to detect these compounds based on their biological activity, quantification by spectrophotometry or high-performance liquid chromatography (HPLC ) relies on their physiochemical properties and is more reliable.

IL-37 exerts therapeutic effects in experimental autoimmune encephalomyelitis through the receptor complex IL-1R5/IL-1R8.

Background: Interleukin 37 (IL-37), a member of IL-1 family, broadly suppresses inflammation in many pathological conditions by acting as a dual-function cytokine in that IL-37 signals via the extracellular receptor complex IL1-R5/IL-1R8, but it can also translocate to the nucleus. However, whether IL-37 exerts beneficial actions in neuroinflammatory diseases, such as multiple sclerosis, remains to be elucidated. Thus, the goals of the present study were to evaluate the therapeutic effects of IL-37 in a mouse model of multiple sclerosis, and if so, whether this is mediated via the extracellular receptor complex IL-1R5/IL-1R8. Methods: We used a murine model of MS, the experimental autoimmune encephalomyelitis (EAE). We induced EAE in three different single and double transgenic mice (hIL-37tg, IL-1R8 KO, hIL-37tg-IL-1R8 KO) and wild type littermates. We also induced EAE in C57Bl/6 mice and treated them with various forms of recombinant human IL-37 protein. Functional and histological techniques were used to assess locomotor deficits and demyelination. Luminex and flow cytometry analysis were done to assess the protein levels of pro-inflammatory cytokines and different immune cell populations, respectively. qPCRs were done to assess the expression of IL-37, IL-1R5 and IL-1R8 in the spinal cord of EAE, and in blood peripheral mononuclear cells and brain tissue samples of MS patients. Results: We demonstrate that IL-37 reduces inflammation and protects against neurological deficits and myelin loss in EAE mice by acting via IL1-R5/IL1-R8. We also reveal that administration of recombinant human IL-37 exerts therapeutic actions in EAE mice. We finally show that IL-37 transcripts are not up-regulated in peripheral blood mononuclear cells and in brain lesions of MS patients, despite the IL-1R5/IL-1R8 receptor complex is expressed. Conclusions: This study presents novel data indicating that IL-37 exerts therapeutic effects in EAE by acting through the extracellular receptor complex IL-1R5/IL-1R8, and that this protective physiological mechanism is defective in MS individuals. IL-37 may therefore represent a novel therapeutic avenue for the treatment of MS with great promising potential.

Extracellular and nuclear roles of IL-37 after spinal cord injury.

Interleukin 37 (IL-37) is an anti-inflammatory cytokine of the interleukin 1 family. Transgenic mice expressing the human form of the IL37 gene (hIL-37Tg) display protective effects in several animal models of disease. Previous data from our group revealed that IL-37 limits inflammation after spinal cord injury (SCI) and ameliorates tissue damage and functional deficits. IL-37 can exert its anti-inflammatory effects by translocating to the nucleus or acting as an extracellular cytokine. However, whether this protection after SCI is mediated by translocating to the nucleus, activating of extracellular receptors, or both, is currently unknown. In the present study, we used different transgenic animals to answer this question. We demonstrated that the beneficial effects of IL-37 on functional and histological outcomes after SCI were lost in the lack of the extracellular receptor IL-1R8, indicating that IL-37 induces protection as an extracellular cytokine. On the other hand, transgenic mice with the nuclear function of IL-37 abolished (hIL-37D20ATg) showed significant improvement in locomotor skills and myelin sparing after SCI, indicating that nuclear pathway is not required for the protective actions of IL-37. Moreover, we also showed that the therapeutic effects of the recombinant IL-37 protein are produced only in the presence of the extracellular receptor IL-1R8, further highlighting the importance of the extracellular function of this cytokine after SCI. Finally, we revealed that the administration of recombinant IL-37 protein exerted therapeutic actions when administered in the lesion site but not systemically. This work demonstrated for the first time that translocation of IL-37 to the nucleus is not required for the beneficial actions of this cytokine after SCI and highlights the importance of the extracellular signaling of IL-37 to mediate neuroprotective actions.

Structural and Bioactivity Characterization of Filipin Derivatives from Engineered Streptomyces filipinensis Strains Reveals Clues for Reduced Haemolytic Action.

The rise in the number of immunocompromised patients has led to an increased incidence of fungal infections, with high rates of morbidity and mortality. Furthermore, misuse of antifungals has boosted the number of resistant strains to these agents; thus, there is urgent need for new drugs against these infections. Here, the in vitro antifungal activity of filipin III metabolic intermediates has been characterized against a battery of opportunistic pathogenic fungi-Candida albicans, Candida glabrata, Candida krusei, Cryptococcus neoformans, Trichosporon cutaneum, Trichosporon asahii, Aspergillus nidulans, Aspergillus niger, and Aspergillus fumigatus-using the Clinical and Laboratory Standards Institute broth microdilution method. Structural characterization of these compounds was undertaken by mass spectrometry (MS) and nuclear magnetic resonance (NMR) following HPLC purification. Complete NMR assignments were obtained for the first time for filipins I and II. In vitro haemolytic assays revealed that the haemolytic action of these compounds relies largely on the presence of a hydroxyl function at C26, since derivatives lacking such moiety show remarkably reduced activity. Two of these derivatives, 1'-hydroxyfilipin I and filipin I, show decreased toxicity towards cholesterol-containing membranes while retaining potent antifungal activity, and could constitute excellent leads for the development of efficient pharmaceuticals, particularly against Cryptococcosis.

The Streptomyces filipinensis Gamma-Butyrolactone System Reveals Novel Clues for Understanding the Control of Secondary Metabolism.

Streptomyces γ-butyrolactones (GBLs) are quorum sensing communication signals triggering antibiotic production. The GBL system of Streptomyces filipinensis, the producer of the antifungal agent filipin, has been investigated. Inactivation of sfbR (for S. filipinensis γ-butyrolactone receptor), a GBL receptor, resulted in a strong decrease in production of filipin, and deletion of sfbR2, a pseudo-receptor, boosted it, in agreement with lower and higher levels of transcription of filipin biosynthetic genes, respectively. It is noteworthy that none of the mutations affected growth or morphological development. While no ARE (autoregulatory element)-like sequences were found in the promoters of filipin genes, suggesting indirect control of production, five ARE sequences were found in five genes of the GBL cluster, whose transcription has been shown to be controlled by both S. filipinensis SfbR and SfbR2. In vitro binding of recombinant SfbR and SfbR2 to such sequences indicated that such control is direct. Transcription start points were identified by 5' rapid amplification of cDNA ends, and precise binding regions were investigated by the use of DNase I protection studies. Binding of both regulators took place in the promoter of target genes and at the same sites. Information content analysis of protected sequences in target promoters yielded an 18-nucleotide consensus ARE sequence. Quantitative transcriptional analyses revealed that both regulators are self-regulated and that each represses the transcription of the other as well as that of the remaining target genes. Unlike other GBL receptor homologues, SfbR activates its own transcription whereas SfbR2 has a canonical autorepression profile. Additionally, SfbR2 was found here to bind the antifungal antimycin A as a way to modulate its DNA-binding activity.IMPORTANCEStreptomyces GBLs are important signaling molecules that trigger antibiotic production in a quorum sensing-dependent manner. We have characterized the GBL system from S. filipinensis, finding that two key players of this system, the GBL receptor and the pseudo-receptor, each counteracts the transcription of the other for the modulation of filipin production and that such control over antifungal production involves an indirect effect on the transcription of filipin biosynthetic genes. Additionally, the two regulators bind the same sites, are self-regulated, and repress the transcription of three other genes of the GBL cluster, including that encoding the GBL synthase. In contrast to all the GBL receptors known, SfbR activates its own synthesis. Moreover, the pseudo-receptor was identified as the receptor of antimycin A, thus extending the range of examples supporting the idea of signaling effects of antibiotics in Streptomyces The intricate regulatory network depicted here should provide important clues for understanding the regulatory mechanism governing secondary metabolism.