Macrophages and Galectin 3 Control Bacterial Burden in Acute and Subacute Murine Leptospirosis That Determines Chronic Kidney Fibrosis

Previous studies have suggested that macrophages may contribute to acute Leptospira dissemination, as well as having a major role in kidney fibrosis. Our aim was to characterize the role of macrophages and galectin 3 (Gal-3) on the survival, clinical course, bacterial burden, interstitial nephritis, and chronic kidney fibrosis in Leptospira interrogans serovar Copenhageni (LIC)-induced experimental murine leptospirosis. C57BL/6J mice depleted of macrophages by liposome-encapsulated clodronate treatment and infected with LIC presented a higher bacterial burden, had reduced subacute nephritis and enhanced chronic kidney fibrosis relative to untreated, infected mice. Moreover, LIC infection in mice whose Gal-3 was disrupted (Lgals3-/-) had a higher bacterial burden and enhanced subacute nephritis and chronic kidney fibrosis when compared to C57BL/6J wild-type mice. Chronic fibrosis did not correlate with higher transcription levels of TGF-β1 or IL-13 in the kidneys. Kidney fibrosis was found in chronically infected rats as well as in wild infected rats. On the other hand, human fibroblast cultures exhibited enhanced differentiation to myofibroblasts after treatment with LIC. Our results demonstrate that macrophages and Gal-3 play a critical role in controlling the LIC burden but has a minor role in subsequent fibrosis. Instead, kidney fibrosis was better correlated with bacterial burden. Taken together, our results do not support a role for macrophages to disseminate leptospires during acute infection, nor in chronic kidney fibrosis.Fil: Ferrer, Maria Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Scharrig Fernandez, Maria Emilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Charó, Nancy Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Rípodas, Ana L.. Bio-lab; ArgentinaFil: Drut, Ricardo. Provincia de Buenos Aires. Ministerio de Salud. Hospital de Niños "Sor María Ludovica" de La Plata; ArgentinaFil: Carrera Silva, Eugenio Antonio. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Nagel, Ariel Gastón. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; ArgentinaFil: Nally, Jarlath E.. United States Department of Agriculture. Agriculture Research Service; Estados UnidosFil: Montes de Oca, Daniela Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Ecología, Genética y Evolución de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Ecología, Genética y Evolución de Buenos Aires; ArgentinaFil: Schattner, Mirta Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Gomez, Ricardo Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; Argentin

STXBP2-R190C Variant in a Patient With Neonatal Hemophagocytic Lymphohistiocytosis (HLH) and G6PD Deficiency Reveals a Critical Role of STXBP2 Domain 2 on Granule Exocytosis

Neonatal hemophagocytic lymphohistiocytosis (HLH) is a medical emergency that can be associated with significant morbidity and mortality. Often these patients present with familial HLH (f-HLH), which is caused by gene mutations interfering with the cytolytic pathway of cytotoxic T-lymphocytes (CTLs) and natural killer cells. Here we describe a male newborn who met the HLH diagnostic criteria, presented with profound cholestasis, and carried a maternally inherited heterozygous mutation in syntaxin-binding protein-2 [STXBP2, c.568C\u3eT (p.Arg190Cys)] in addition to a severe pathogenic variant in glucose 6-phosphate dehydrogenase [G6PD, hemizygous c.1153T\u3eC (Cys385Arg)]. Although mutations in STXBP2 gene are associated with f-HLH type 5, the clinical and biological relevance of the p.Arg190Cys mutation identified in this patient was uncertain. To assess its role in disease pathogenesis, we performed functional assays and biochemical and microscopic studies. We found that p.Arg190Cys mutation did not alter the expression or subcellular localization of STXBP2 or STX11, neither impaired the STXBP2/STX11 interaction. In contrast, forced expression of the mutated protein into normal CTLs strongly inhibited degranulation and reduced the cytolytic activity outcompeting the effect of endogenous wild-type STXBP2. Interestingly, arginine 190 is located in a structurally conserved region of STXBP2 where other f-HLH-5 mutations have been identified. Collectively, data strongly suggest that STXBP2-R190C is a deleterious variant that may act in a dominant-negative manner by probably stabilizing non-productive interactions between STXBP2/STX11 complex and other still unknown factors such as the membrane surface or Munc13-4 protein and thus impairing the release of cytolytic granules. In addition to the contribution of STXBP2-R190C to f-HLH, the accompanied G6PD mutation may have compounded the clinical symptoms; however, the extent by which G6PD deficiency has contributed to HLH in our patient remains unclear

Macrophages and Galectin 3 Control Bacterial Burden in Acute and Subacute Murine Leptospirosis That Determines Chronic Kidney Fibrosis

Previous studies have suggested that macrophages may contribute to acute Leptospira dissemination, as well as having a major role in kidney fibrosis. Our aim was to characterize the role of macrophages and galectin 3 (Gal-3) on the survival, clinical course, bacterial burden, interstitial nephritis, and chronic kidney fibrosis in Leptospira interrogans serovar Copenhageni (LIC)-induced experimental murine leptospirosis. C57BL/6J mice depleted of macrophages by liposome-encapsulated clodronate treatment and infected with LIC presented a higher bacterial burden, had reduced subacute nephritis and enhanced chronic kidney fibrosis relative to untreated, infected mice. Moreover, LIC infection in mice whose Gal-3 was disrupted (Lgals3-/-) had a higher bacterial burden and enhanced subacute nephritis and chronic kidney fibrosis when compared to C57BL/6J wild-type mice. Chronic fibrosis did not correlate with higher transcription levels of TGF-β1 or IL-13 in the kidneys. Kidney fibrosis was found in chronically infected rats as well as in wild infected rats. On the other hand, human fibroblast cultures exhibited enhanced differentiation to myofibroblasts after treatment with LIC. Our results demonstrate that macrophages and Gal-3 play a critical role in controlling the LIC burden but has a minor role in subsequent fibrosis. Instead, kidney fibrosis was better correlated with bacterial burden. Taken together, our results do not support a role for macrophages to disseminate leptospires during acute infection, nor in chronic kidney fibrosis.Facultad de Ciencias Médica

Forebrain mapping of secretoneurin-like immunoreactivity and its colocalization with isotocin in the preoptic nucleus and pituitary gland of goldfish.

International audienceSecretoneurin, a 33-34 amino acid neuropeptide derived from the proteolytic processing of the secretogranin-II precursor protein, is reasonably well conserved in evolution. Goldfish secretoneurin shares >75% similarity overall with other vertebrate secretoneurin sequences. The secretoneurin peptide has numerous functions that include neuroinflammation, neurotransmitter release, and neuroendocrine regulation. A detailed description of the central distribution of secretoneurin immunoreactivity is only known for the rat. Using our polyclonal antibody against the central, conserved core of the secretoneurin peptide we studied the distribution of secretoneurin-like immunoreactivity in the goldfish brain. Secretoneurin immunoreactivity was found in the olfactory bulb, entopeduncular nucleus, preoptic nucleus, lateral part of the lateral tuberal nucleus, posterior periventricular nucleus, nucleus of the posterior recess, the nucleus of the saccus vasculosus, and nucleus isthmi. Secretoneurin-immunoreactive fibers were found in the dorsal part of the dorsal telencephalon, ventral and lateral parts of the ventral telencephalon, periventricular preoptic nucleus, pituitary, and the ventrocaudal aspect of the nucleus of the lateral recess. The most conspicuous secretoneurin immunoreactivity was found in the magnocellular and parvocellular cells of the preoptic nucleus that project to the pituitary. Double-labeling studies indicated coexpression with isotocin, the fish homolog of mammalian oxytocin. Clear colabeling for secretoneurin and isotocin in fibers terminating in the neurointermediate lobe suggests that secretoneurin maybe coreleased with isotocin. Previous work indicates that secretoneurin stimulates the release of luteinizing hormone from the goldfish anterior pituitary. Our findings further support a reproductive role for secretoneurin and related peptides, given the importance of oxytocin family peptides in reproductive behavior in vertebrates

Interaction of Leptospira with the Innate Immune System

International audienceInnate immunity encompasses immediate host responses that detect and respond to microbes. Besides recognition by the complement system (see the chapter by A. Barbosa, this volume), innate immunity concerns cellular responses. These are triggered through recognition of conserved microbial components (called MAMPs) by pattern recognition receptors (PRRs), leading, through secretion of cytokines, antimicrobial peptides, and immune mediators, to cellular recruitment and phagocytosis. Leptospira spp. are successful zoonotic pathogenic bacteria that obviously overcome the immune system of their hosts. The first part of this chapter summarizes what is known about leptospires recognition and interaction with phagocytes and other innate immune cells, and the second part describes specific interactions of leptospiral MAMPs with PRRs from the TLR and NLR families. On the one hand, pathogenic leptospires appear to escape macrophage and neutrophil phagocytosis. On the other hand, studies about PRR sensing of leptospires remain very limited, but suggest that pathogenic leptospires escape some of the PRRs in a host-specific manner, due to peculiar cell wall specificities or post-translational modifications that may impair their recognition. Further studies are necessary to clarify the mechanisms and consequences of leptospiral escape on phagocytic functions and hopefully give clues to potential therapeutic strategies aimed at restoring the defective activation of PRRs by pathogenic Leptospira spp

Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its ‘Minimal Information for Studies of Extracellular Vesicles’, which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly