Phagocytosis Of Pathogens Research Articles

Intracellular and extracellular effector activity of mouse neutrophils in response to cutaneous and visceral Leishmania parasites

Neutrophils are short-lived phagocytic cells equipped with several receptors for pathogen recognition and phagocytosis and have intracellular and extracellular effector mechanisms that can inactivate pathogens. Leishmaniases are diseases caused by different species of Leishmania that mainly afflicts poorer populations of tropical and subtropical regions and immunocompromised individuals. Thus, the present study aims to investigate the effector response of murine neutrophils to species of Leishmania causing American cutaneous leishmaniasis and zoonotic visceral leishmaniasis by evaluating pattern recognition receptors (PRR) and intracellular and extracellular effector microbicide activity. When exposed to Leishmania parasites, mouse neutrophils produced superoxide, released enzymes in the extracellular space and generated neutrophil extracellular traps, although PRR gene expression is negatively regulated. L. infantum, L. guyanensis, and L. shawi inhibited enzymatic activity, whereas L. amazonensis reduced the emission of extracellular structures. These findings indicate that although neutrophils trigger several microbicide mechanisms, Leishmania parasites can manipulate extracellular effector mechanisms. The present study also provides evidence that neutrophils can internalize parasites by coiling phagocytosis.

FEVER AND RESPIRATORY DISTRESS IN AN INFANT: MANNOSE-BINDING LECTIN DEFICIENCY?

Introduction Mannose-binding lectin (MBL) plays an important role in innate immunity by mediating phagocytosis and complement-mediated destruction of pathogens. Decreased MBL levels have been associated with an increased susceptibility to herpes simplex virus, influenza A, Pseudomonas aeruginosa, and Staphylococcus aureus. MBL- deficient individuals typically present with upper respiratory tract infections, acute otitis media, or lupus-like symptoms. We present an infant with multiple infections who was diagnosed with MBL deficiency. Case Description This is a 7-week-old term male with a history of failure to thrive and acute otitis media. He was admitted with fever, respiratory distress, and new onset seizures. A respiratory panel was positive for Influenza B, Enterovirus, and Rhinovirus. Physical exam was significant for hepatomegaly and a desquamating rash on his lips, buttocks, and left hand consistent with possible Staphylococcus scalded skin syndrome, treated with antibiotics. An immunodeficiency was suspected because of multiple infections. Laboratory work-up revealed a positive blood culture to MRSA and low mannose-binding lectin levels (initial 11.9 ng/mL, repeat 3 ng/mL - normal range >50 ng/mL). Lymphocyte subsets (T, B and NK cells), quantitative immunoglobulins (IgA, IgM, and IgG), and CH50 were all normal. He was diagnosed with MBL deficiency. Discussion MBL deficiency is a poorly understood disorder of the innate immune system. There have been limited reports of MBL deficiency despite a high prevalence in certain populations. This case highlights the importance of having innate immune system defects in the differential diagnosis for early diagnosis and management of patients with a history of multiple infections. Mannose-binding lectin (MBL) plays an important role in innate immunity by mediating phagocytosis and complement-mediated destruction of pathogens. Decreased MBL levels have been associated with an increased susceptibility to herpes simplex virus, influenza A, Pseudomonas aeruginosa, and Staphylococcus aureus. MBL- deficient individuals typically present with upper respiratory tract infections, acute otitis media, or lupus-like symptoms. We present an infant with multiple infections who was diagnosed with MBL deficiency. This is a 7-week-old term male with a history of failure to thrive and acute otitis media. He was admitted with fever, respiratory distress, and new onset seizures. A respiratory panel was positive for Influenza B, Enterovirus, and Rhinovirus. Physical exam was significant for hepatomegaly and a desquamating rash on his lips, buttocks, and left hand consistent with possible Staphylococcus scalded skin syndrome, treated with antibiotics. An immunodeficiency was suspected because of multiple infections. Laboratory work-up revealed a positive blood culture to MRSA and low mannose-binding lectin levels (initial 11.9 ng/mL, repeat 3 ng/mL - normal range >50 ng/mL). Lymphocyte subsets (T, B and NK cells), quantitative immunoglobulins (IgA, IgM, and IgG), and CH50 were all normal. He was diagnosed with MBL deficiency. MBL deficiency is a poorly understood disorder of the innate immune system. There have been limited reports of MBL deficiency despite a high prevalence in certain populations. This case highlights the importance of having innate immune system defects in the differential diagnosis for early diagnosis and management of patients with a history of multiple infections.

245 - Thioredoxin as a marker for inflammatory macrophages

Monocyte-derived macrophages are immune cells derived from hematopoietic progenitors. They interact with a variety of environmental stimuli and carry out highly variable functions, from pathogen phagocytosis to wound healing. Macrophages have traditionally been classified as M1 or M2 based on their mode of activation; this framework corresponds to proinflammatory and anti-inflammatory states, respectively. We hypothesized that these inflammatory states are associated with altered metabolic or redox environments within the cell. To test this hypothesis, human monocyte derived macrophages differentiated using granulocyte-monocyte colony stimulating factor (GMCSF) or monocyte colony stimulating factor (MCSF), which are known to respectively produce pro-inflammatory and anti-inflammatory macrophages, were analyzed by scRNAseq. This allowed an unbiased interrogation of cellular state while accounting for potential cell-to-cell heterogeneity. Highly expressed genes distinguishing GMCSF-differentiated macrophages from MCSF-differentiated macrophages included thioredoxin 1, peroxidredoxin 1, and peroxiredoxin 3. We validated the increased expression of thioredoxin 1 on the protein level by western blot. MCSF-differentiated macrophages classically activated with IFNg and LPS also showed increased expression of thioredoxin, indicating that high thioredoxin expression is a conserved feature of an inflammatory macrophage state.

The Role of Collectins and Galectins in Lung Innate Immune Defense.

Different families of endogenous lectins use complementary defense strategies against pathogens. They may recognize non-self glycans typically found on pathogens and/or host glycans. The collectin and galectin families are prominent examples of these two lectin categories. Collectins are C-type lectins that contain a carbohydrate recognition domain and a collagen-like domain. Members of this group include surfactant protein A (SP-A) and D (SP-D), secreted by the alveolar epithelium to the alveolar fluid. Lung collectins bind to several microorganisms, which results in pathogen aggregation and/or killing, and enhances phagocytosis of pathogens by alveolar macrophages. Moreover, SP-A and SP-D influence macrophage responses, contributing to resolution of inflammation, and SP-A is essential for tissue-repair functions of macrophages. Galectins also function by interacting directly with pathogens or by modulating the immune system in response to the infection. Direct binding may result in enhanced or impaired infection of target cells, or can have microbicidal effects. Immunomodulatory effects of galectins include recruitment of immune cells to the site of infection, promotion of neutrophil function, and stimulation of the bactericidal activity of infected macrophages. Moreover, intracellular galectins can serve as danger receptors, promoting autophagy of the invading pathogen. This review will focus on the role of collectins and galectins in pathogen clearance and immune response activation in infectious diseases of the respiratory system.

Decidual stromal cell-derived PGE2 regulates macrophage responses to microbial threat.

Bacterial chorioamnionitis causes adverse pregnancy outcomes, yet host-microbial interactions are not well characterized within gestational membranes. The decidua, the outermost region of the membranes, is a potential point of entry for bacteria ascending from the vagina to cause chorioamnionitis. We sought to determine whether paracrine communication between decidual stromal cells and macrophages shaped immune responses to microbial sensing. Decidual cell-macrophage interactions were modeled in vitro utilizing decidualized, telomerase-immortalized human endometrial stromal cells (dTHESCs) and phorbol ester-differentiated THP-1 macrophage-like cells. The production of inflammatory mediators in response to LPS was monitored by ELISA for both cell types, while phagocytosis of bacterial pathogens (Escherichia coli and Group B Streptococcus (GBS)) was measured in THP-1 cells or primary human placental macrophages. Diclofenac, a non-selective cyclooxygenase inhibitor, and prostaglandin E2 (PGE2 ) were utilized to interrogate prostaglandins as decidual cell-derived paracrine immunomodulators. A mouse model of ascending chorioamnionitis caused by GBS was utilized to assess the colocalization of bacteria and macrophages in vivo and assess PGE2 production. In response to LPS, dTHESC and THP-1 coculture demonstrated enhancement of most inflammatory mediators, but a potent suppression of macrophage TNF-α generation was observed. This appeared to reflect a paracrine-mediated effect of decidual cell-derived PGE2 . In mice with GBS chorioamnionitis, macrophages accumulated at sites of bacterial invasion with increased PGE2 in amniotic fluid, suggesting such paracrine effects might hold relevance in vivo. These data suggest key roles for decidual stromal cells in modulating tissue responses to microbial threat through release of PGE2 .

Benzo(a)pyrene attenuates the pattern-recognition-receptor induced proinflammatory phenotype of murine macrophages by inducing IL-10 expression in an aryl hydrocarbon receptor-dependent manner

Polycyclic aromatic hydrocarbons such as benzo(a)pyrene (BaP) are environmental contaminants known to be immunosuppressive. Most effects of BaP towards immune cells are thought to be mediated through activation of the aryl hydrocarbon receptor (AhR). The AhR is a ligand-activated transcription factor, which plays a critical modulatory role in various cells during immune response. Macrophages are key players in innate immunity against intracellular bacteria and are discussed to be a target of AhR-mediated immune regulation. However, so far there is only incomplete knowledge about the effects of BaP on activated macrophages and whether these effects are AhR-dependent in each case. Using murine bone marrow-derived macrophages (BMMs) stimulated with heat-killed salmonellae as a source of different pathogen-associated molecular patterns (PAMPs) for stimulation of different pattern recognition receptors (PRRs) as an in-vitro model, we studied the immunomodulatory effects of low-dose BaP exposure. PRR-activated BMMs produced nitric oxide (NO) and a spectrum of proinflammatory cytokines, i.e. tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and IL-12 but also the anti-inflammatory cytokine IL-10. While BaP exposure suppressed the production of proinflammatory cytokines, the secretion of IL-10 was augmented. Moreover, BaP exposure increased the expression of major histocompatibility complex class II (MHC-II), CD14, Fcγ receptor I (FcγRI/CD64), or CD86, enhanced NO production and phagocytosis what may be beneficial for phagocytosis and killing of microbial pathogens. Of note, without PRR activation low-dose BaP exposure has little influence on the macrophage phenotype. BMMs from AhR-deficient (Ahr−/−) mice were widely refractory to BaP-induced modulation of cytokine production, surface marker expression, and functional properties in response to PAMPs stimulation, indicating that these effects are dependent on AhR. In summary, these data suggest that induction of AhR-mediated signalling pathways by BaP may attenuate the proinflammatory phenotype of PRR-activated BMMs, while activating IL-10-mediated anti-inflammatory properties but also enhancing uptake and killing of pathogens as well as antigen presentation. Together these features imply a favourable role of BaP exposure for macrophage functions in an ongoing immune response. However, the strong induction of IL-10 may lead to defective pathogen clearance and subsequently to chronic persistent infection. This concept suggests an inhibitory rather than a supporting influence of environmental BaP on immunity to infection or cancer and also emphasises the important regulatory role of AhR in immunity and inflammation.

Progress in innate immunity-related genes in insects

Insects have evolved a strong innate immune system to defense pathogens and adverse conditions. Insect innate immune system comprises of humoral immunity and cellular immunity. Humoral immunity mainly includes three signaling pathways, i.e., Toll, IMD and JAK/STAT, by which signal transduction and immune pathways regulate expression of immune-related genes and activate antimicrobial peptides and other effectors. Cellular immunity contains phagocytosis, encapsulation and nodulation of pathogens, which is mediated by hemolymph cells. In recent years, with the rapid development of insect genomics, a large number of immune-related genes have been characterized from insect genome data using bioinformatic methods. Studies on these genes have significantly deepened the understanding of molecular mechanisms of insect innate immune system. Insect immune-related genes can be divided into seven categories by gene functions: recognition, signaling transduction, modulator, effectors, melanization, RNA interference and other genes. The humoral immunity and cellular immunity are regulated by the interactions among these immune-related genes. In this review, we summarize the classification, function and evolution of insect immune-related genes, and propose future research directions of insect innate immunity, which will be helpful for understanding molecular mechanisms of insect innate immune system and developing new strategies for controlling pest insects.

Binding capacity of mannose-binding lectin (MBL) is associated with the severity of chronic Chagas cardiomyopathy

Chagas disease (CD) is a global problem. Currently, it affects approximately 15 million individuals in Latin America. It is well know that the human immune response is related to different clinical manifestations. Mannose binding lectin (MBL) plays an important role in innate immunity, and it mediates the phagocytosis and complement-mediated destruction of pathogens. The binding capacity is enhanced by the oligomerization of MBL. In this study, we evaluated the serum concentration and the binding capacity of MBL in patients with chronic chagasic cardiomyopathy. A total of 77 patients with chronic CD were included with indeterminate (n = 19), mild cardiac (n = 29) and severe cardiac (n = 29) forms. The serum concentration and the binding capacity were measured using enzyme-linked immunosorbent assays (ELISA). There was no significant difference in the serum MBL levels between the groups of patients. However, we found a relationship between the binding capacity and the groups studied. Our results suggest that binding capacity of MBL could be an indicator of clinical manifestation in Chronic Chagas cardiomyopathy. Furthermore, combined with the Mannose Binding Index results in a useful clinical tool for management of Chronic Chagas Patients.

Structural studies on ligand binding ability of Siglec-2 using molecular modeling techniques

Siglecs are attractive therapeutic target of the major homologous subfamily of I-type lectins. The primary role of Siglecs may actually lie in the recognition and phagocytosis of bacterial pathogens that express sialic acids, maintenance of myelin organization, and inhibition of neurite outgrowth, cell-cell interactions between neurons and glial cells etc. Siglec-2, a member of the Siglec family expressed on the surface of maturing B cells and B cell lymphomas and regulates signal transduction. In this work, 3-D structure of human Siglec-2 was predicted using molecular modeling techniques. The structure of the complex in solution of Siglec-2 with ligand, 6′-Sialyl-N-acetyl lactose (6′-SialylLacNAc) was predicted using a novel docking technique. The structural analysis of the complex and calculation of theoretical dissociation constant value will help to ascertain functional roles of such sugar binding protein.

Neutrophil Activation During Septic Shock.

In addition to their well-known role as the cellular mediators of immunity, key other roles have been identified for neutrophils during septic shock. Importantly, neutrophils indeed play a critical role in the recently described immunothrombosis concept and in septic shock-induced coagulopathy. Septic shock is one of the most severe forms of infection, characterized by an inadequate host response to the pathogenic organism. This host response involves numerous defense mechanisms with an intense cellular activation, including neutrophil activation. Neutrophils are key cells of innate immunity through complex interactions with vascular cells and their activation may participate in systemic tissue damages. Their activation also leads to the emission of neutrophil extracellular traps, which take part in both pathogen circumscription and phagocytosis, but also in coagulation activation. Neutrophils thus stand at the interface between hemostasis and immunity, called immunothrombosis.The present review will develop a cellular approach of septic shock pathophysiology focusing on neutrophils as key players of septic shock-induced vascular cell dysfunction and of the host response, associating immunity and hemostasis. We will therefore first develop the role of neutrophils in the interplay between innate and adaptive immunity, and will then highlight recent advances in our understanding of immunothrombosis septic shock-induced coagulopathy.

Role of heme oxygenase-1 in potentiation of phagocytic activity of macrophages by taurine chloramine: Implications for the resolution of zymosan A-induced murine peritonitis

Phagocytosis of pathogens by macrophages is crucial for the successful resolution of inflammation induced by microbial infection. Taurine chloramine (TauCl), an endogenous anti-inflammatory and antioxidative substance, is produced by reaction between taurine and hypochlorous acid by myeloperoxidase activity in neutrophils under inflammatory conditions. In the present study, we investigated the effect of TauCl on resolution of acute inflammation caused by fungal infection using a zymosan A-induced murine peritonitis model. TauCl administration reduced the number of the total peritoneal leukocytes, while it increased the number of peritoneal monocytes. Furthermore, TauCl promoted clearance of pathogens remaining in the inflammatory environment by macrophages. When the macrophages isolated from thioglycollate-treated mice were treated with TauCl, their phagocytic capability was enhanced. In the murine macrophage-like RAW264.7 cells treated with TauCl, the proportion of macrophages clearing the zymosan A particles was also increased. TauCl administration resulted in elevated expression of heme oxygenase-1 (HO-1) in the peritoneal macrophages. Pharmacologic inhibition of HO-1 activity or knockdown of HO-1 in the murine macrophage RAW264.7 cells abolished the TauCl-induced phagocytosis, whereas the overexpression of HO-1 augmented the phagocytic ability of macrophages. Moreover, peritoneal macrophages isolated from HO-1 null mice failed to mediate TauCl-induced phagocytosis. Our results suggest that TauCl potentiates phagocytic activity of macrophages through upregulation of HO-1 expression.

Size-Dependent Organelle Transport during Phagocytosis

Phagocytosis of bacteria and other pathogens by macrophages is a key process of the mammalian immune system. The intracellular maturation of phagosomes which often leads to the degradation of the internalized pathogens shows high organelle-to-organelle variations that are not clearly understood. An important part of the maturation is the phagosomal transport from the cell periphery towards the perinuclear region. We hypothesize that the phagosome size influences the phagosomal transport and therefore also potentially the maturation process. We tested this hypothesis by tracking phagosomes with different diameters between 1 µm and 3 µm inside macrophages. We show that the transport efficiency increases with increasing phagosome size although the instantaneous velocities of the investigated phagosomes are very similar to each other. In addition, we found that the bi-directional motion as well as the transport from the nucleus back to the periphery decreases with increasing phagosome size. We furthermore show that dynein is significantly involved in the phagosomal transport, in particular in the persistent centripetal transport of large phagosomes. In addition, we found that actin-dependent motion is also contributing to the transport, in particular to the transport of small phagosomes. Furthermore we investigated the spatial distribution of dyneins and microtubules, and found that density differences between the nucleus-facing side of phagosomes and the opposite side can explain part of the observed transport characteristics. Our findings suggest that a basic size-dependent cellular sorting mechanism might exist that supports inward transport of large phagocytosed pathogens for facilitating their digestion and that simultaneously supports outward transport of small pathogen fragments for example for antigen presentation.

Apoptotic Cell Clearance in Drosophila melanogaster.

The swift clearance of apoptotic cells (ACs) (efferocytosis) by phagocytes is a critical event during development of all multicellular organisms. It is achieved through phagocytosis by professional or amateur phagocytes. Failure in this process can lead to the development of inflammatory autoimmune or neurodegenerative diseases. AC clearance has been conserved throughout evolution, although many details in its mechanisms remain to be explored. It has been studied in the context of mammalian macrophages, and in the nematode Caenorhabditis elegans, which lacks “professional” phagocytes such as macrophages, but in which other cell types can engulf apoptotic corpses. In Drosophila melanogaster, ACs are engulfed by macrophages, glial, and epithelial cells. Drosophila macrophages perform similar functions to those of mammalian macrophages. They are professional phagocytes that participate in phagocytosis of ACs and pathogens. Study of AC clearance in Drosophila has identified some key elements, like the receptors Croquemort and Draper, promoting Drosophila as a suitable model to genetically dissect this process. In this review, we survey recent works of AC clearance pathways in Drosophila, and discuss the physiological outcomes and consequences of this process.

Conversion of NOX2 into a constitutive enzyme in vitro and in living cells, after its binding with a chimera of the regulatory subunits

During the phagocytosis of pathogens by phagocyte cells, the NADPH oxidase complex is activated to produce superoxide anion, a precursor of microbial oxidants. The activated NADPH oxidase complex from phagocytes consists in two transmembrane proteins (Nox2 and p22phox) and four cytosolic proteins (p40phox, p47phox, p67phox and Rac1-2). In the resting state of the cells, these proteins are dispersed in the cytosol, the membrane of granules and the plasma membrane. In order to synchronize the assembly of the cytosolic subunits on the membrane components of the oxidase, a fusion of the cytosolic proteins p47phox, p67phox and Rac1 named trimera was constructed. The trimera investigated in this paper is composed of the p47phox segment 1–286, the p67phox segment 1–212 and the mutated Rac1(Q61L). We demonstrate that the complex trimera-cyt b558 is functionally comparable to the one containing the separated subunits. Each of the subunits p47phox, p67phox and Rac1Q61L has kept its own activating property. The trimera is produced in an activated conformation as seen by circular dichroism. However, the presence of amphiphile is still necessary in a cell-free system to trigger superoxide anion production. The COS7gp91-p22 cells expressing the trimera produce continuously superoxide anion at high rate. This constitutive activity in cells can be of particular interest for understanding the NADPH oxidase functioning independently of signaling pathways.

SNX10 promotes phagosome maturation in macrophages and protects mice against Listeria monocytogenes infection.

Listeria monocytogenes (L. monocytogenes), which is a facultative intracellular bacterial pathogen that causes listeriosis, is widely used to study the mammalian immune response to infection. After phagocytosis by professional phagocytes, L. monocytogenes is initially contained within phagosomes, which mature into phagolysosomes, where the bacteria are degraded. Although phagocytosis and subsequent phagosome maturation is essential for the clearance of infectious microbial pathogens, the underlying regulatory mechanisms are still unclear. SNX10 (Sorting nexin 10) has the simplest structure of the SNX family and has been reported to regulate endosomal morphology, which might be crucial for macrophage function, including phagocytosis and digestion of pathogens, inflammatory response, and antigen presentation. Our results showed that SNX10 expression was upregulated following L. monocytogenes infection in macrophages. It was also revealed that SNX10 promoted phagosome maturation by recruiting the Mon1-Ccz1 complex to endosomes and phagosomes. As a result, SNX10 deficiency decreased the bacterial killing ability of macrophages, and SNX10-deficient mice showed increased susceptibility to L. monocytogenes infection in vivo. Thus, this study revealed an essential role of SNX10 in controlling bacterial infection.

Synthetic immunostimulatory glycans interference with host cell apoptosis upon of Toxoplasma gondii infection, in vitro

Toxoplasmosis is a protozoan infection of humans and animals caused by Toxoplasma gondii, and it’s continuous public health and food safety issue. The tachyzoites (Tg) of T. gondii are the most important stage, as they come in direct contact with immune cells such as a macrophage. Tg can modulate and prevent apoptosis of immune cells while promoting survival of the pathogen. Infections caused by Tg can be eradicated if immune cells could stimulate apoptosis and kill pathogens upon exposure. Apoptosis is characterized by the release of mediators, namely Caspases (Cas). New means are required for inducing apoptosis and enhance immunity in the infected host cell to control toxoplasmosis. The present study investigated whether Synthetic Immuno-stimulatory Glycans (SIGs) influence Cas and Nitric oxide (NO) release and led to Tg damage. Galβ1-3Gal-PAA-fluor (SIG1), Fucα1-4GlcNAcβ-PAA-fluor (SIG2) and GlcNAcβ1-3GalNAcα-PAA-fluor (SIG3) constituted samples studied principally. Murine macrophage had been exposed to the Tg then the SIGs effects on Cas and NO production were determined after 20 hours of pathogen phagocytosis. Here we report that the SIGs had potent in vitro activity against T. gondii; SIG2 was more effective than SIG1 and SIG3, representative by SIG2 treated infected macrophages can induced infected macrophages to release Cas1, 3, and 9. Maximum production of NO by infected macrophages was noticed following the expoxure to all SIGs. Therefore the present study provided the method for the selection of SIGs ligands bearing immunostimulatory factor and apoptotic stimuli properties.

The RNA-seq analysis suggests a potential multi-component complement system in oyster Crassostrea gigas

The complement system is one of the major effector mechanisms of immune system, playing essential roles in both the innate and adaptive immune responses. In the present study, the counterparts of vertebrate complement components were identified by screening the sequenced genome of Crassostrea gigas, resulting in the identification of 792 gene models containing complement-related domains. The transcriptome of haemocytes at 6, 12 and 24 h post lipopolysaccharides (LPS) stimulation showed differential expression of 77 C1q domain containing proteins, 53 C-type lectins and 42 fibrinogen-related proteins. mRNAs encoding 18 serine protease domain-containing (SPC) proteins, 4 MACPF-domain containing proteins and 11 C3 receptor-like proteins were up-regulated upon LPS stimulation, and CgC3 mRNA was significantly increased at 12 h. The presence of CgC3 was confirmed in cell free plasma and was present in three subunit chains as expected for the processed mature protein. The complement related PRRs with coiled coil regions and SPC proteins with CUB domains may function in the activation of CgC3, whereas, the C3-like receptors with integrin-α/β domain mediated the phagocytosis of C3-labled pathogens. These PRRs appear to serve as opsonins to promote phagocytosis of opsonized pathogens. The overall results suggested the existence of a potential multi-component complement system in C. gigas.

SMN deficiency negatively impacts red pulp macrophages and spleen development in mouse models of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a progressive neurodegenerative disease that is the leading genetic cause of infantile death. It is caused by a severe deficiency of the ubiquitously expressed Survival Motor Neuron (SMN) protein. SMA is characterized by α-lower motor neuron loss and muscle atrophy, however, there is a growing list of tissues impacted by a SMN deficiency beyond motor neurons. The non-neuronal defects are observed in the most severe Type I SMA patients and most of the widely used SMA mouse models, however, as effective therapeutics are developed, it is unclear whether additional symptoms will be uncovered in longer lived patients. Recently, the immune system and inflammation has been identified as a contributor to neurodegenerative diseases such as ALS. To determine whether the immune system is comprised in SMA, we analyzed the spleen and immunological components in SMA mice. In this report, we identify: a significant reduction in spleen size in multiple SMA mouse models and a pathological reduction in red pulp and extramedullary hematopoiesis. Additionally, red pulp macrophages, a discrete subset of yolk sac-derived macrophages, were found to be altered in SMA spleens even in pre-symptomatic post-natal day 2 animals. These cells, which are involved in iron metabolism and the phagocytosis of erythrocytes and blood-borne pathogens are significantly reduced prior to the development of the neurodegenerative hallmarks of SMA, implying a differential role of SMN in myeloid cell ontogeny. Collectively, these results demonstrate that SMN deficiency impacts spleen development and suggests a potential role for immunological development in SMA.

Inhibition of WAVE Regulatory Complex Activation by a Bacterial Virulence Effector Counteracts Pathogen Phagocytosis

SummaryTo establish pathogenicity, bacteria must evade phagocytosis directed by remodeling of the actin cytoskeleton. We show that macrophages facilitate pathogen phagocytosis through actin polymerization mediated by the WAVE regulatory complex (WRC), small GTPases Arf and Rac1, and the Arf1 activator ARNO. To establish extracellular infections, enteropathogenic (EPEC) and enterohaemorrhagic (EHEC) Escherichia coli hijack the actin cytoskeleton by injecting virulence effectors into the host cell. Here, we find that the virulence effector EspG counteracts WRC-dependent phagocytosis, enabling EPEC and EHEC to remain extracellular. By reconstituting membrane-associated actin polymerization, we find that EspG disabled WRC activation through two mechanisms: EspG interaction with Arf6 blocked signaling to ARNO while EspG binding of Arf1 impeded collaboration with Rac1, thereby inhibiting WRC recruitment and activation. Investigating the mode of EspG interference revealed sites in Arf1 required for WRC activation and a mechanism facilitating pathogen evasion of innate host defenses.

Characterization of neutrophils and macrophages from ex vivo-cultured murine bone marrow for morphologic maturation and functional responses by imaging flow cytometry

Neutrophils and macrophages differentiate from common myeloid progenitors in the bone marrow, where they undergo nuclear morphologic changes during maturation. During this process, both cell types acquire critical innate immune functions that include phagocytosis of pathogens, and for neutrophils the release of nuclear material called nuclear extracellular traps (NETs). Primary cells used to study these functions are typically purified from mature mouse tissues, but bone marrow-derived ex vivo cultures provide more abundant numbers of progenitors and functionally mature cells. Routine analyses of these cells use conventional microscopy and flow cytometry, which present limitations; microscopy is laborious and subjective, whereas flow cytometry lacks spatial resolution. Here we describe methods to generate enriched populations of neutrophils or macrophages from cryopreserved mouse bone marrow cultured ex vivo, and to use imaging flow cytometry that combines the resolution of microscopy with flow cytometry to analyze cells for morphologic features, phagocytosis, and NETosis.