Microanatomy and ultrastructure of kidney interstitial cells and nephron in brown trout (Salmo trutta Linnaeus, 1758) at different stages of the life cycle

The study focuses on the microanatomy and ultrastructural changes in the trunk kidney interstitium cells and nephrons in parr, smolt and spawning brown trout Salmo trutta Linnaeus, 1758 sampled in Luga River and Solka River, the tributaries of the Baltic Sea. Regardless of the type of cells or their structure, there were changes in their areas and the number and structure of organelles responsible for the transport, synthetic and energetic function of cells. Our data on the morphology of the nephron combined with data on its physiology suggest a fundamental change in kidney function during the parr-smolt transformation before migration; this could be a preadaptation for a successful life in saltwater where urine output is sharply reduced. Thus, detected structural features of the trunk kidney in brown trout S. trutta are cytological markers of the migration process. The numbers of lymphocytes, neutrophils and eosinophils with segmented nuclei increased from parr to smolts and then to spawners; only monotypic specific granules in neutrophils were found in smolts and spawners. Cells with radially arranged vesicles were described for the first time in brown trout S. trutta renal interstitium. Their origin has not yet been established. The shape of these cells changed from spherical to trihedral during fish maturation. All the above ultrastructural changes of renal interstitium cells could be considered cytological markers of cell maturity.

Galectin-9 Triggers Neutrophil-Mediated Anticancer Immunity

In earlier studies, galectin-9 (Gal-9) was identified as a multifaceted player in both adaptive and innate immunity. Further, Gal-9 had direct cytotoxic and tumor-selective activity towards cancer cell lines of various origins. In the current study, we identified that treatment with Gal-9 triggered pronounced membrane alterations in cancer cells. Specifically, phosphatidyl serine (PS) was rapidly externalized, and the anti-phagocytic regulator, CD47, was downregulated within minutes. In line with this, treatment of mixed neutrophil/tumor cell cultures with Gal-9 triggered trogocytosis and augmented antibody-dependent cellular phagocytosis of cancer cells. Interestingly, this pro-trogocytic effect was also due to the Gal-9-mediated activation of neutrophils with upregulation of adhesion markers and mobilization of gelatinase, secretory, and specific granules. These activation events were accompanied by a decrease in cancer cell adhesion in mixed cultures of leukocytes and cancer cells. Further, prominent cytotoxicity was detected when leukocytes were mixed with pre-adhered cancer cells, which was abrogated when neutrophils were depleted. Taken together, Gal-9 treatment potently activated neutrophil-mediated anticancer immunity, resulting in the elimination of epithelial cancer cells.

Neutrophil specific granule and NETosis defects in gray platelet syndrome

Gray platelet syndrome (GPS) is an autosomal recessive bleeding disorder characterized by a lack of α-granules in platelets and progressive myelofibrosis. Rare loss-of-function variants in neurobeachin-like 2 (NBEAL2), a member of the family of beige and Chédiak-Higashi (BEACH) genes, are causal of GPS. It is suggested that BEACH domain containing proteins are involved in fusion, fission, and trafficking of vesicles and granules. Studies in knockout mice suggest that NBEAL2 may control the formation and retention of granules in neutrophils. We found that neutrophils obtained from the peripheral blood from 13 patients with GPS have a normal distribution of azurophilic granules but show a deficiency of specific granules (SGs), as confirmed by immunoelectron microscopy and mass spectrometry proteomics analyses. CD34+ hematopoietic stem cells (HSCs) from patients with GPS differentiated into mature neutrophils also lacked NBEAL2 expression but showed similar SG protein expression as control cells. This is indicative of normal granulopoiesis in GPS and identifies NBEAL2 as a potentially important regulator of granule release. Patient neutrophil functions, including production of reactive oxygen species, chemotaxis, and killing of bacteria and fungi, were intact. NETosis was absent in circulating GPS neutrophils. Lack of NETosis is suggested to be independent of NBEAL2 expression but associated with SG defects instead, as indicated by comparison with HSC-derived neutrophils. Since patients with GPS do not excessively suffer from infections, the consequence of the reduced SG content and lack of NETosis for innate immunity remains to be explored.

Eosinophils and Neutrophils—Molecular Differences Revealed by Spontaneous Raman, CARS and Fluorescence Microscopy

Leukocytes are a part of the immune system that plays an important role in the host’s defense against viral, bacterial, and fungal infections. Among the human leukocytes, two granulocytes, neutrophils (Ne) and eosinophils (EOS) play an important role in the innate immune system. For that purpose, eosinophils and neutrophils contain specific granules containing protoporphyrin-type proteins such as eosinophil peroxidase (EPO) and myeloperoxidase (MPO), respectively, which contribute directly to their anti-infection activity. Since both proteins are structurally and functionally different, they could potentially be a marker of both cells’ types. To prove this hypothesis, UV−Vis absorption spectroscopy and Raman imaging were applied to analyze EPO and MPO and their content in leukocytes isolated from the whole blood. Moreover, leukocytes can contain lipidic structures, called lipid bodies (LBs), which are linked to the regulation of immune responses and are considered to be a marker of cell inflammation. In this work, we showed how to determine the number of LBs in two types of granulocytes, EOS and Ne, using fluorescence and coherent anti-Stokes Raman scattering (CARS) microscopy. Spectroscopic differences of EPO and MPO can be used to identify these cells in blood samples, while the detection of LBs can indicate the cell inflammation process.

Acetylation of C/EBPε is a prerequisite for terminal neutrophil differentiation

Key Points C/EBPε acetylation regulates C/EBPε transcriptional activity. C/EBPε acetylation is required for neutrophil differentiation and the formation of neutrophil-specific granules.

miRNA-130a Regulates C/EBP-ε During Granulopoiesis.

Abstract 2137 Granulopoiesis is a tightly regulated process. A strictly regulated temporal expression of different transcription factors is crucial for proper neutrophil development. Dysregulation of transcription factors may result in impaired antimicrobial function of the neutrophils and may cause leukemia. microRNAs (miRNAs) are small noncoding RNAs, that post-transcriptionally regulate protein expression. Dysregulation of miRNA expression has been shown to contribute to the development of neoplastic myeloid diseases such as myelodysplastic syndrome (MDS), and acute myeloid leukemia (AML) and point to an important role of miRNAs in fine tuning of protein regulation in granulopoiesis. We found that miR-130a is highly expressed in immature proliferating granulocytic precursors, and has a low expression in more mature cells in granulopoiesis. Using microRNA target-prediction software we found the transcription factor C/EBP-ε to be a putative target for miR-130a. C/EBP-ε is essential for expression of the genes encoding the proteins stored in the specific granules of the neutrophil. C/EBP-ε also play an important role for cessation of cell division at the myelocyte stage and the further progress of differentiation. The biological effect of lacking C/EBP-ε can be observed in the C/EBP-ε knock-out mouse, with accumulation of immature granulocytic precursors in the bone marrow and a severely reduced number of terminally differentiated granulocytes in both bone marrow and peripheral blood. The anti-microbial defense is impaired in the C/EBP-ε knock-out mice which are significantly more susceptible to infection than wild-type mice. We demonstrate that the mRNA for C/EBP-epsilon is present already in the late promyelocytes while the protein does not appear until the myelocyte stage. This indicates a posttranscriptional regulation of C/EBP-epsilon expression e.g. by microRNAs. In-silico analysis has shown that the 3′-UTR of the C/EBP-epsilon mRNA contains one binding site for miR-130a. We have verified this by analysis of luciferase reporter constructs carrying the 3′-UTR of C/EBP-ε with the miR-130a binding-site. The repressive effect of miR-130a was abolished by point mutations in the miRNA–binding site. Next we analyzed a murine granulocytic cell line (MPRO), which stably over expressed miR-130a, and found a reduction of the protein level of C/EBP-ε. We also found a significant decrease in the expression of different genes encoding proteins stored in specific granules such as lactoferrin and hCAP18. Interestingly, we also found elevated expression of MPO mRNA in these cells indicating a more immature phenotype of the cells. Finally, we transiently transfected myelocytes and metamyelocytes from human bone marrow with pre-miR-130a and demonstrated that this has a significant effect on C/EBP-ε protein levels also in human cells. The reverse result was obtained when transfecting the cells with an anti-miR-130a oligonucleotide. These results indicate that miR-130a plays an important regulatory role in granulopoiesis and if dysregulated, could lead to a compromised immune function and developmental progression through its regulation of C/EBP-ε. Grant acknowledgment: The Danish Cancer Society, Lundbeck foundation, Brøchner Mortensen foundation, Medical Research Council. Disclosures: No relevant conflicts of interest to declare.

A1AT In Human Neutrophil Granulocytes.

Abstract 3784 Alpha-1-antitrypsin (A1AT) is an important inhibitor of the neutrophil serine proteases elastase, cathepsin G, and proteinase 3. A1AT is produced mainly by the liver and secreted to plasma. A1AT deficiency caused by the PiZZ mutation in the A1AT gene leads to accumulation of mutated A1AT in the liver which may induce liver cell necrosis and necessitate liver transplantation. In a recently performed profiling of mRNA expression during terminal neutrophil differentiation in the bone marrow, we found that A1AT mRNA increases from the promyelocyte stage and up, indicating that A1AT is a constituent of all neutrophil granules. We examined the localization and production of A1AT in healthy donor neutrophils and investigated whether the structure or function of neutrophils is affected in individuals with A1AT deficiency. RT- PCR for A1AT performed on neutrophil precursors isolated from normal human bone marrow showed that the mRNA level is highly upregulated as the cells mature in the bone marrow and even increases further as the cells enter the blood stream. Biosynthesis studies revealed that A1AT is produced by all stages of neutrophil maturation in the bone marrow and is efficiently retained in the cells as judged by pulse chase studies. Neutrophils from circulating blood also produce A1AT but this is not retained in the cells. Stimulation of neutrophils from peripheral blood with G-CSF during 24 hours resulted in a 20 fold increase in A1AT biosynthesis which was largely released to the medium. Subcellullar fractionation of blood neutrophils on a 4-layer Percoll density gradient revealed 3 forms of A1AT. A doublet band at 37 and 44 kD both with immunoreactivity against A1AT was observed in fractions corresponding to azurophil granules (cf biosynthesis of this form in promyelocytes). A band with mw of 52 kD, corresponding to the form present in blood plasma, was observed in fractions that contain NGAL, a marker of specific granules and in fractions that contain gelatinase. The 52 kD band was also observed in fractions containing albumin as expected, since secretory vesicles contain plasma proteins. The localization of A1at in neutrophil granules was further confirmed by exocytosis studies. Neutrophils were stimulated with PMA which mobilizes secretory vesicles and gelatinase granules efficiently and approximately 50% of specific granules. Only the 52 mw form of A1AT was released from cells during stimulation while none of the 37/44 double band was released. This is in agreement with localization of this double band in azurophil granules and with localization of the 52 kD form in specific granules, gelatinase granules and secretory vesicles. In addition, a high molecular weight form of A1AT was observed at 76 kD corresponding to the mw of A1AT complexed with neutrophil elastase. We isolated neutrophils from patients with the ZZ genotype of A1AT deficiency which had either been liver transplanted or lung transplanted. The neutrophils were examined by electron microscopy for detection of structural abnormalities and by exocytosis studies for detection of functional abnormalities. Electron micrographs did not reveal any abnormality in neutrophil structure and in none of the neutrophils examined (from 6 patients) did we observe abnormal granules akin to the intracellular accumulation of A1AT in liver cells from patients. We did, however, observe reduction in the total intracellular amount of A1AT in neutrophils from patients that had been lung transplanted but not in neutrophils from liver transplanted patients. This most likely reflects that secretory vesicles of neutrophils from lung transplanted will not contain A1AT as this is still severely deficient in plasma from lung transplanted patients, while liver transplanted patients will have normal levels of A1AT in plasma and hence take up normal amounts into their secretory vesicles. Release of granule proteins in response to stimulation by fMLP or PMA did not reveal any functional abnormality in neutrophils from A1AT deficient patients. Based on these studies we conclude that the A1AT deficiency does not inflict functional or structural abnormalities on neutrophils, and suggest that A1AT generated and released from neutrophils may contribute to anti-protease defense in tissues. Disclosures: No relevant conflicts of interest to declare.

Olfactomedin 4, An Indicator of the Existence of Neutrophil Subsets.

Abstract 3775 Olfactomedin 4 (OLFM4) was initially identified as a gene highly induced in myeloid stem cells by G-CSF treatment and independently as a gene highly expressed in colon cancers. OLFM4 was predicted in a bioinformatics analysis as associated with neutrophil specific granules. We analyzed the expression of OLFM4 mRNA in myeloid cells from normal human bone marrow and demonstrated that expression of OLFM4 mRNA is similar to the expression of LCN2 which codes for the specific granule protein NGAL (Figure 1), but distinct from expression of mRNA for myeloperoxidase and gelatinase which are marker proteins for azurophil granules and gelatinase granules, respectively. Subcellular fractionation of peripheral blood neutrophils demonstrated complete co-localization of OLFM4 with NGAL, and stimulation of neutrophils with fMLP or PMA resulted in co-release of NGAL and OLFM4, indirectly proving that OLFM4 is a genuine constituent of neutrophil specific granules. Figure 1. mRNA expression profiles for OLFM4 and LCN2 in populations enriched in myeloblasts/promyelocytes (MB/PM), myelocytes/metamyelocytes (MY/MM), banded cells/segmented cells (BC) and peripheral blood neutrophils (pb-PMN) normalized to ACTB. Figure 1. mRNA expression profiles for OLFM4 and LCN2 in populations enriched in myeloblasts/promyelocytes (MB/PM), myelocytes/metamyelocytes (MY/MM), banded cells/segmented cells (BC) and peripheral blood neutrophils (pb-PMN) normalized to ACTB. Interestingly, immunohistochemistry showed OLFM4 expression in only a subset of neutrophils (figure 2). We suspected that this might be dependent on the antibody, but two different commercial antibodies and an in-house antibody raised against a synthetic OLFM4 derived peptide, all polyclonal, showed similar patterns. Flow cytometry confirmed the existence of two populations of neutrophils, one expressing OLFM4 the other not. Figure 2. Immunohistochemistry of OLFM4 in neutrophils. Figure 2. Immunohistochemistry of OLFM4 in neutrophils. Immunohistochemistry of bone marrow cells showed that OLFM4 appears in myelocytes and is maintained in the cells during further maturation of the cells to segmented neutrophils. Again, only 30% of the neutrophil precursors from bone marrow stain positive for OLFM4 indicating, that different subsets of human neutrophils may exist. Disclosures: No relevant conflicts of interest to declare.