Jennifer L. Johns

Extramedullary Hematopoiesis: A New Look at the Underlying Stem Cell Niche, Theories of Development, and Occurrence in Animals

Extramedullary hematopoiesis (EMH) is the formation and development of blood cells outside the medullary spaces of the bone marrow. Although widely considered an epiphenomenon, secondary to underlying primary disease and lacking serious clinical or diagnostic implications, the presence of EMH is far from incidental on a molecular basis; rather, it reflects a well-choreographed suite of changes involving stem cells and their microenvironment (the stem cell niche). The goals of this review are to reconsider the molecular basis of EMH based on current knowledge of stem cell niches and to examine its role in the pathophysiologic mechanisms of EMH in animals. The ability of blood cells to home, proliferate, and mature in extramedullary tissues of adult animals reflects embryonic patterns of hematopoiesis and establishment or reactivation of a stem cell niche. This involves pathophysiologic alterations in hematopoietic stem cells, extracellular matrix, stromal cells, and local and systemic chemokines. Four major theories involving changes in stem cells and/or their microenvironment can explain the development of most occurrences of EMH: (1) severe bone marrow failure; (2) myelostimulation; (3) tissue inflammation, injury, and repair; and (4) abnormal chemokine production. EMH has also been reported within many types of neoplasms. Understanding the concepts and factors involved in stem cell niches enhances our understanding of the occurrence of EMH in animals and its relationship to underlying disease. In turn, a better understanding of the prevalence and distribution of EMH in animals and its molecular basis could further inform our understanding of the hematopoietic stem cell niche.

Avian Hematology and Related Disorders

Hematology is an essential component of veterinary practice. The interpretation of avian blood cells provides many challenges. Practitioners must be able to recognize normal morphology and function of cells to interpret changes in those cells. This article describes the normal morphology of avian erythrocytes, leukocytes, and thrombocytes. Changes observed in erythrocytes and leukocytes during disease and major differential diagnoses are discussed. A brief overview of avian blood parasites is also presented.

Infection with Anaplasma phagocytophilum induces multilineage alterations in hematopoietic progenitor cells and peripheral blood cells

ABSTRACT Infection with Anaplasma phagocytophilum, a gram-negative, lipopolysaccharide (LPS)-negative, obligate intracellular bacterium, results in multiple peripheral blood cytopenias. We hypothesized that infection with this organism would result in decreased bone marrow (BM) function and shifts in hematopoietic progenitor cells (HPCs) and lineage-committed cells in a well-established murine model of infection. HPCs and lineage-committed progenitors were enumerated in the BM and spleen during acute infection. BM cytokine production and BM CXCL12 expression were determined. Infection resulted in peripheral blood bicytopenia, marked decreases in the number of lineage-committed HPCs in the BM along with concurrent increases in the number of lineage-committed HPCs in the spleen, and a mixed, predominantly myelosuppressive BM cytokine environment. There was significant downregulation of CXCL12 in BM cells that may have been partially responsible for changes in HPC trafficking observed. Changes occurred in the absence of direct pathogen infection of BM cells. Hematopoietic lineage assessment demonstrated that there was loss of erythrocytes and B lymphocytes from the BM along with increased granulopoiesis. These changes were accompanied by splenomegaly due to lymphoid hyperplasia and increased hematopoiesis, most notably erythropoiesis. These changes largely mimic well-described inflammation and endotoxin-mediated effects on the BM and spleen; however, the numbers of peripheral blood neutrophils appear to be independently modulated as granulocytic hyperplasia does not result in neutrophilia. Our findings highlight a well-conserved series of events that we demonstrate can be instigated by an LPS-negative pathogen in the absence of an endotoxin-mediated acute proinflammatory response.

Anaplasma phagocytophilum Dihydrolipoamide Dehydrogenase 1 Affects Host-Derived Immunopathology during Microbial Colonization

ABSTRACT Anaplasma phagocytophilum is a tick-borne rickettsial pathogen that provokes an acute inflammatory response during mammalian infection. The illness caused by A. phagocytophilum, human granulocytic anaplasmosis, occurs irrespective of pathogen load and results instead from host-derived immunopathology. Thus, characterizing A. phagocytophilum genes that affect the inflammatory process is critical for understanding disease etiology. By using an A. phagocytophilum Himar1 transposon mutant library, we showed that a single transposon insertion into the A. phagocytophilum dihydrolipoamide dehydrogenase 1 gene (lpda1 [APH_0065]) affects inflammation during infection. A. phagocytophilum lacking lpda1 revealed enlargement of the spleen, increased splenic extramedullary hematopoiesis, and altered clinicopathological abnormalities during mammalian colonization. Furthermore, LPDA1-derived immunopathology was independent of neutrophil infection and correlated with enhanced reactive oxygen species from NADPH oxidase and nuclear factor (NF)-κB signaling in macrophages. Taken together, these findings suggest the presence of different signaling pathways in neutrophils and macrophages during A. phagocytophilum invasion and highlight the importance of LPDA1 as an immunopathological molecule.

The Systemic Immune State of Super-shedder Mice Is Characterized by a Unique Neutrophil-dependent Blunting of TH1 Responses

Host-to-host transmission of a pathogen ensures its successful propagation and maintenance within a host population. A striking feature of disease transmission is the heterogeneity in host infectiousness. It has been proposed that within a host population, 20% of the infected hosts, termed super-shedders, are responsible for 80% of disease transmission. However, very little is known about the immune state of these super-shedders. In this study, we used the model organism Salmonella enterica serovar Typhimurium, an important cause of disease in humans and animal hosts, to study the immune state of super-shedders. Compared to moderate shedders, super-shedder mice had an active inflammatory response in both the gastrointestinal tract and the spleen but a dampened TH1 response specific to the secondary lymphoid organs. Spleens from super-shedder mice had higher numbers of neutrophils, and a dampened T cell response, characterized by higher levels of regulatory T cells (Tregs), fewer T-bet+ (TH1) T cells as well as blunted cytokine responsiveness. Administration of the cytokine granulocyte colony stimulating factor (G-CSF) and subsequent neutrophilia was sufficient to induce the super-shedder immune phenotype in moderate-shedder mice. Similar to super-shedders, these G-CSF-treated moderate-shedders had a dampened TH1 response with fewer T-bet+ T cells and a loss of cytokine responsiveness. Additionally, G-CSF treatment inhibited IL-2-mediated TH1 expansion. Finally, depletion of neutrophils led to an increase in the number of T-bet+ TH1 cells and restored their ability to respond to IL-2. Taken together, we demonstrate a novel role for neutrophils in blunting IL-2-mediated proliferation of the TH1 immune response in the spleens of mice that are colonized by high levels of S. Typhimurium in the gastrointestinal tract.

Use of an in vitro biotinylation technique for determination of posttransfusion survival of fresh and stored autologous red blood cells in Thoroughbreds

OBJECTIVE To evaluate N-hydroxysuccinimide (NHS)-biotin labeling of equine RBCs and determine posttransfusion survival of autologous equine RBCs stored in citrate phosphate dextrose adenine-1 (CPDA-1) for 0, 1, 14, and 28 days. ANIMALS 13 healthy adult Thoroughbreds. PROCEDURES Serial dilutions of biotin and streptavidin-phycoerythrin (PE) were evaluated in vitro in blood collected from 3 horses. One horse was used to determine RBC distribution and recovery. Twelve horses were allocated to 4 groups for in vivo experiments in which blood was collected into CPDA-1. Blood was labeled with biotin and reinfused or stored at 4 degrees C for 1, 14, or 28 days prior to labeling with NHS-biotin and reinfusion. Posttransfusion blood samples were collected 15 minutes and 1, 2, 3, 5, 7, 14, 21, 28, and 35 days after reinfusion. Biotin-labeled RBCs were detected via flow cytometry by use of streptavidin-PE. Posttransfusion lifespan of RBCs and RBC half-life were determined. RESULTS Optimal biotin concentration was 0.04 pg of biotin/RBC, and the optimal streptavidin-PE ratio was 1.2 microg of streptavidin-PE/1 x 10(6) RBCs. Posttransfusion lifespan of autologous RBCs was 99, 89, 66, and 59 days after storage for 0, 1, 14, and 28 days, respectively. Storage did not result in significant alterations in RBC lifespan. Mean posttransfusion RBC half-life was 50, 45, 33, and 29 days for 0, 1, 14, and 28 days of storage, respectively. CONCLUSIONS AND CLINICAL RELEVANCE Biotin can be used to label equine RBCs for RBC survival studies. Posttransfusion survival of equine autologous RBCs was greater than previously reported.

Post-transfusion survival of biotin-labeled allogeneic RBCs in adult horses.

BACKGROUND Post-transfusion survival of allogeneic RBCs has been reported to be much shorter in horses than in other species. We hypothesized that post-transfusion survival of biotinylated allogeneic equine RBCs would be greater than the survival previously reported from studies using radioactive RBC-labeling techniques. OBJECTIVE The study objective was to determine post-transfusion survival of N-hydroxysuccinimide (NHS)-biotin-labeled allogeneic equine RBCs transfused into adult horses. METHODS Horses were adults and included 5 donors and 5 recipients. All horses were blood-typed, and donors were paired with recipients based upon blood type and crossmatch results. Donor blood was collected in a volume of 4 L into citrate phosphate dextrose adenine-1 and stored for 24 hours, labeled with NHS-biotin, and re-infused into recipients. Post-transfusion blood samples were collected at 15 minutes and at 1, 2, 3, 5, 7, 14, 21, 28, and 35 days. Biotin-labeled RBCs were detected by flow cytometry using streptavidin-phycoerythrin. Post-transfusion survival at 24 hours, lifespan, and half-life of biotinylated RBCs were determined. RESULTS Mean ± SD survival of biotinylated RBCs at 24 hours post-transfusion was 95 ± 24%; the mean lifespan of transfused allogeneic RBCs was 39 days based on calculation of a linear regression survival curve, and mean post-transfusion RBC half-life was 20 days. CONCLUSIONS Post-transfusion survival of 24-hour stored equine allogeneic RBCs was greater than previously reported but less than that observed for other companion animal species. Mechanisms for the relatively short post-transfusion lifespan of allogeneic equine RBCs remain unknown and warrant further study.

Anaplasma phagocytophilum and Ehrlichia muris induce cytopenias and global defects in hematopoiesis

Peripheral blood cytopenias result from a wide variety of infectious diseases and may significantly complicate the prognosis and outcome of those diseases. Infection with Gram-negative, LPS-negative members of the Anaplasmataceae family, including Anaplasma phagocytophilum and Ehrlichia muris, results in bi- or pancytopenia. The specific mechanisms responsible for the cytopenias are unknown. Viruses and bacteria have profound yet often discrepant effects on bone marrow (BM) and circulating numbers of blood cells. Multiple line-age cytopenias (bi- and pancytopenia) are a frequent sequel of viral infections [1]. Different viruses can directly infect BM stromal cells or haematopoietic stem cells and/or modulate cytokine production by accessory cells. Infections with Gram-negative, LPS-positive bacteria result in an endotoxin-mediated, acute phase, proinflammatory cascade of events that result in granulocyte efflux from the BM, peripheral neutrophilia and BM hyperplasia [2]. In contrast, gram-negative, LPS-negative bacteria, including members of the Anaplasmataceae family, elicit a very different, poorly-characterised, cascade of events. Haematologic alterations in both natural and experimental infection include a marked thrombocytopenia, moderate leukopenia (lymphopenia and neutropenia) and mild to moderate, non-regenerative anaemia [3,4]. Cytopenias vary from profound and irreversible (for example, dogs with chronic E. canis infection) to moderate and transient. Paradoxically, although Anaplasma family organisms display cell tropism for specific haematopoietic cell lineages, infection, regardless of aetiologic agent, host species or cell target, is characterised by bicytopenia or, occasionally, pancytopenia. This suggests the potential for more global haematopoietic disturbances. We hypothesised that the cytopenias may be due in part to bacteria-induced haematopoietic suppression. C57/Bl6 mice were infected intraperitoneally with E. muris and samples were collected on days 0–60 post-infection. C3H/HeN mice were infested with A. phagocytophilum-infected Ixodes scapularis nymphal ticks and sampled on days 0–21 post-infection. Blood samples were collected for a complete blood cell analysis and quantitative PCR for pathogen detection (A. phagocytophilum p44 DNA). BM was collected for haematopoietic colony-forming assays, cytology, histology and quantitative PCR. Cultured BM supernatants from A. phagocytophilum infected mice were collected for cytokine analysis. Thrombocytopenia and anaemia (bicytopenia) developed in both E. muris and A. phagocytophilum-infected mice. Leukopenia was variable. A. phagocytophilum infection induced significant decreases in BM proliferation and differentiation from day 4 to day 21 post-infection, concurrent with the cytopenias (Fig. 1). E. muris infection resulted in a similarly dramatic decrease in BM proliferation and differentiation on days 8 to 14 post-infection. Decreases in BM haematopoietic production included all lineages. Haematopoietic colony forming assays are supplemented with cytokines and assess haematopoietic stem cell/progenitor cell activity in the absence of stromal cells. As such, findings support infection-induced alterations in stem cell differentiation, proliferation or mobilisation. Both thrombocytopenia and myelosuppression were more severe in mice infected with A. phagocytophilum via tick bite compared with mice infected intraperitoneally in previous studies. Thrombocytopenia did not correlate with pathogen burden in blood, as assessed by quantitative PCR, but was strongly correlated with BM colony numbers in A. phagocytophilum-infected mice. This temporal association suggests that rapid changes in BM may be contributing to alterations in circulating platelet numbers. Cytologic evaluation of BM revealed infection-induced lymphocyte depletion and a reciprocal granulocytic hyperplasia in both infection models, similar to kinetics noted in other models of inflammation [5]. In addition, A. phagocytophilum infection resulted in BM production of myelosuppressive chemokines, including the murine interleukin-8 homologues, macrophage inflammatory protein-2 and KC. Findings are compatible with previous work that demonstrated a predominance of myelosuppressive chemokines in human BM after A. phagocytophilum infection [6]. Anaplasma family pathogens appear to perturb normal haematopoiesis, resulting in shifts in BM cell populations and transient failure of haematopoietic colony formation, which may contribute to peripheral blood cytopenias. Fig. 1 Bone marrow was collected from Anaplasma phagocytophilum-infected and uninfected mice on days 0–21 after tick-borne infection. An aliquot of BM was plated in duplicate and cultured in complete MethoCult media for 7–10 days (StemCell Technologies) ...

Activation of the nuclear factor-κB pathway during postnatal lung inflammation preserves alveolarization by suppressing macrophage inflammatory protein-2

A significant portion of lung development is completed postnatally during alveolarization, rendering the immature lung vulnerable to inflammatory stimuli that can disrupt lung structure and function. Although the NF-κB pathway has well-recognized pro-inflammatory functions, novel anti-inflammatory and developmental roles for NF-κB have recently been described. Thus, to determine how NF-κB modulates alveolarization during inflammation, we exposed postnatal day 6 mice to vehicle (PBS), systemic lipopolysaccharide (LPS), or the combination of LPS and the global NF-κB pathway inhibitor BAY 11-7082 (LPS + BAY). LPS impaired alveolarization, decreased lung cell proliferation, and reduced epithelial growth factor expression. BAY exaggerated these detrimental effects of LPS, further suppressing proliferation and disrupting pulmonary angiogenesis, an essential component of alveolarization. The more severe pathology induced by LPS + BAY was associated with marked increases in lung and plasma levels of macrophage inflammatory protein-2 (MIP-2). Experiments using primary neonatal pulmonary endothelial cells (PEC) demonstrated that MIP-2 directly impaired neonatal PEC migration in vitro; and neutralization of MIP-2 in vivo preserved lung cell proliferation and pulmonary angiogenesis and prevented the more severe alveolar disruption induced by the combined treatment of LPS + BAY. Taken together, these studies demonstrate a key anti-inflammatory function of the NF-κB pathway in the early alveolar lung that functions to mitigate the detrimental effects of inflammation on pulmonary angiogenesis and alveolarization. Furthermore, these data suggest that neutralization of MIP-2 may represent a novel therapeutic target that could be beneficial in preserving lung growth in premature infants exposed to inflammatory stress.

Prevalence of Batrachochytrium dendrobatidis in 120 archived specimens of Lithobates catesbeianus (American bullfrog) collected in California, 1924-2007.

The chytrid fungus, Batrachochytrium dendrobatidis (Bd), has been identified as a major cause of the recent worldwide amphibian decline. Numerous species in North America alone are under threat or have succumbed to Bd-driven population extinctions. The American bullfrog (Lithobates catesbeianus) has been reported as a tolerant carrier of Bd. In this report, we used a qPCR assay to test 120 archived American bullfrog specimens collected between 1924 and 2007 in California, USA and Baja California, Mexico. The overall prevalence of Bd infection in this archived population of L. catesbeianus was 19.2%. The earliest positive specimen was collected in Sacramento County, California, USA in 1928 and is to date the earliest positive archived Bd specimen reported globally. These data demonstrate that Bd-infected wild amphibians have been present in California longer than previously known.