Normal Macrophage Function Research Articles

CRISPR Correction of the GBA Mutation in Human-Induced Pluripotent Stem Cells Restores Normal Function to Gaucher Macrophages and Increases Their Susceptibility to Mycobacterium tuberculosis.

Gaucher disease (GD) is an autosomal recessive lysosomal storage disorder caused by mutations in the β-glucocerebrosidase (GCase) GBA gene, which result in macrophage dysfunction. CRISPR (clustered regularly interspaced short palindromic repeats) editing of the homozygous L444P (1448T→C) GBA mutation in type 2 GD (GBA-/-) human-induced pluripotent stem cells (hiPSCs) yielded both heterozygous (GBA+/-) and homozygous (GBA+/+) isogenic lines. Macrophages derived from GBA-/-, GBA+/- and GBA+/+ hiPSCs showed that GBA mutation correction restores normal macrophage functions: GCase activity, motility, and phagocytosis. Furthermore, infection of GBA-/-, GBA+/- and GBA+/+ macrophages with the Mycobacterium tuberculosis H37Rv strain showed that impaired mobility and phagocytic activity were correlated with reduced levels of bacterial engulfment and replication suggesting that GD may be protective against tuberculosis.

Skin Wound Healing: Normal Macrophage Function and Macrophage Dysfunction in Diabetic Wounds

Macrophages play a prominent role in wound healing. In the early stages, they promote inflammation and remove pathogens, wound debris, and cells that have apoptosed. Later in the repair process, they dampen inflammation and secrete factors that regulate the proliferation, differentiation, and migration of keratinocytes, fibroblasts, and endothelial cells, leading to neovascularisation and wound closure. The macrophages that coordinate this repair process are complex: they originate from different sources and have distinct phenotypes with diverse functions that act at various times in the repair process. Macrophages in individuals with diabetes are altered, displaying hyperresponsiveness to inflammatory stimulants and increased secretion of pro-inflammatory cytokines. They also have a reduced ability to phagocytose pathogens and efferocytose cells that have undergone apoptosis. This leads to a reduced capacity to remove pathogens and, as efferocytosis is a trigger for their phenotypic switch, it reduces the number of M2 reparative macrophages in the wound. This can lead to diabetic foot ulcers (DFUs) forming and contributes to their increased risk of not healing and becoming infected, and potentially, amputation. Understanding macrophage dysregulation in DFUs and how these cells might be altered, along with the associated inflammation, will ultimately allow for better therapies that might complement current treatment and increase DFU’s healing rates.

Computational Fine-Tuning of Functional Single Nucleotide Polymorphisms Associated with ACP5 Gene to Characterize Missense Mutations

Acid phosphatase 5 (ACP5) gene plays a vibrant role in the synthesis of a tartrate-resistant acid phosphatase (TRAP5) enzyme. TRAP5 is ~35 KD glycosylated di-iron metalloenzyme responsible for the regulation of osteopontin (protein) activity. There are two isoforms of TRAP5, TRAP5a, and TRAP5b. TRAP5a functions with low enzymatic activity due to a loop interacting with the active site and the more active TRAP5b is generated upon proteolytic cleavage of this loop. TRAP5a works as a marker for systematic macrophage function and chronic inflammation activity, while TRAP5b for osteoclast activity. ACP5 is evolutionarily conserved in nature and acts as a multifunctional protein that involves generations of reactive oxygen species, normal bone development, macrophage function, and osteoblast regulation, affecting a series of pathways, as well as reflecting bone resorption and osteoclast activity. To understand its fundamental role, a functional investigation of missense mutations of the ACP5 gene was carried out through an in-silico approach. Two nsSNPs G109R and L201P were predicted to be deleterious using multiple computational tools like SIFT, Polyphen-1, PolyPhen-2, MAPP, SNAP, Predict SNP and PhD-SNP. Additionally, the structural analysis was performed. The result is that there was no similarity between the native and mutant structures. Therefore, these reported mutations in ACP5 modify the expression, function, and structure of a TRAP5 protein. These findings suggest that TRAP5 can be a therapeutic target in immunological disorders, cancer, and metabolic bone diseases. These deleterious mutations can be lethal to its function and may hamper its therapeutic strategy leading to various diseases such as autoimmune cytopenia, systemic lupus erythematosus (SLE) immune-osseous dysplasia, spasticity with leukodystrophy, moyamoya syndrome, and sjogren's syndrome.

Wild-type huntingtin regulates human macrophage function

The huntingtin (HTT) protein in its mutant form is the cause of the inherited neurodegenerative disorder, Huntington’s disease. Beyond its effects in the central nervous system, disease-associated mutant HTT causes aberrant phenotypes in myeloid-lineage innate immune system cells, namely monocytes and macrophages. Whether the wild-type form of the protein, however, has a role in normal human macrophage function has not been determined. Here, the effects of lowering the expression of wild-type (wt)HTT on the function of primary monocyte-derived macrophages from healthy, non-disease human subjects were examined. This demonstrated a previously undescribed role for wtHTT in maintaining normal macrophage health and function. Lowered wtHTT expression was associated, for instance, with a diminished release of induced cytokines, elevated phagocytosis and increased vulnerability to cellular stress. These may well occur by mechanisms different to that associated with the mutant form of the protein, given an absence of any effect on the intracellular signalling pathway predominantly associated with macrophage dysfunction in Huntington’s disease.

Smad7 is required for normal macrophage function in experimental chronic pancreatitis

Smad7 is required for normal macrophage function in experimental chronic pancreatitis

Tumor-specific macrophage targeting through recognition of retinoid X receptor beta

Macrophages play important and diverse roles during cancer progression. However, cancer therapies based on macrophage modulation are lacking in tools that can recognize and deliver therapeutic payloads to macrophages in a tumor-specific manner. As a result, treatments tend to interfere with normal macrophage functions in healthy organs. We previously identified a macrophage-binding peptide, termed CRV. Here, we show that upon systemic administration into tumor-bearing mice, CRV selectively homes to tumors, extravasates, and preferentially binds to macrophages within. CRV exhibits a higher affinity for tumor macrophages than for other cells in tumors or for other macrophage types elsewhere in the body. We further identified and validated retinoid X receptor beta (RXRB) as the CRV receptor. Intriguingly, although it is known as a nuclear receptor, RXRB shows a prominent cell surface localization that is largely restricted to tumor macrophages. Systemic administration of anti-RXRB antibodies also results in tumor-selective binding to macrophages similar to CRV. Lastly, we demonstrate the ability of CRV to improve the delivery of nano-carriers into solid tumors and macrophages within. In summary, we describe here a novel cell surface marker and targeting tools for tumor macrophages that may aid in future development of macrophage-modulatory cancer therapies.

ACP5: Its Structure, Distribution, Regulation and Novel Functions.

Tartrate-resistant acid phosphatase 5 (ACP5) is an evolutionarily conserved and multifunctional protein that is involved in generations of reactive oxygen species, normal bone development, osteoblast regulation and macrophage function, affecting a series of pathways, as well as reflecting bone resorption and osteoclast activity. Literature searches, systematic reviews and assessments about the structure, distribution, regulation and novel functions of ACP5 were performed in this review from PubMed and Medline databases. Studies demonstrate that RANKL can increase the expression of ACP5 through NFATc1 and c-Fos to accelerate osteoclastogenesis, which also can be regulated by many regulators. Based on the aforementioned information, it is shown that ACP5, together with the phosphatase activity, can medicate the progression and development of human genetic diseases and cancer. As a novel target, ACP5 plays a critical role in preventing, monitoring and treating various kinds of tumors, as well as accelerating the development of a promising therapeutic strategy for human genetic diseases. However, the explicit mechanism between ACP5 and cancer is not so clear. It is necessary and significant for us to pay more in-depth attention.

Pregnancy Induces a Steady-State Shift in Alveolar Macrophage M1/M2 Phenotype That Is Associated With a Heightened Severity of Influenza Virus Infection: Mechanistic Insight Using Mouse Models.

Influenza virus infection during pregnancy is associated with enhanced disease severity. However, the underlying mechanisms are still not fully understood. We hypothesized that normal alveolar macrophage (AM) functions, which are central to maintaining lung immune homeostasis, are altered during pregnancy and that this dysregulation contributes to the increased inflammatory response to influenza virus infection. Time-mated BALB/c mice were infected with a low dose of H1N1 influenza A virus at gestation day 9.5. Inflammatory cells in bronchoalveolar lavage (BAL) fluid were assessed by flow cytometry. Our findings confirm previous reports of increased severity of influenza virus infection in pregnant mice. The heightened inflammatory response detected in BAL fluid from infected pregnant mice was characterized by neutrophil-rich inflammation with concomitantly reduced numbers of AM, which were slower to return to baseline counts, compared with nonpregnant infected mice. The increased infection severity and inflammatory responses to influenza during pregnancy were associated with a pregnancy-induced shift in AM phenotype at homeostatic baseline, from the M1 (ie, classical activation) state toward the M2 (ie, alternative activation) state, as evidence by increased expression of CD301 and reduced levels of CCR7. These results show that pregnancy is associated with an alternatively activated phenotype of AM before infection, which may contribute to heightened disease severity.

Tet2 Deficiency in Macrophages Undermines Heart Repair after Infarction

Background: Clonal hematopoiesis of indeterminate potential (CHIP) is often observed during aging[1]. CHIP is characterized by a clonal expansion of subset blood-cell clones without other hematological abnormalities. Recent studies reported a strong connection between CHIP and cardiovascular disease (CVD). Individuals with mutations in epigenetic regulators (e.g., DNMT3A, TET2, ASXL1 and JAK2) have a higher risk for CVD[2]. In vivo studies using mouse models confirmed that Tet2-deficient HSPCs exhibited clonal expansion. Furthermore, mice transferred with Tet2-deficient HSPCs showed increased risk of atherosclerosis and worse outcome after permanent left anterior descending artery (LAD) ligation induced myocardial infarction (MI)[3,4]. Macrophages were found to be one of the most affected cell types in Tet2 deficient hematopoietic system associated with increased CVD risk[3,4]. Tet2 deficiency in macrophages is known to cause aberrant innate immune response[5]. Tet2 knockout (KO) macrophages provide a protective microenvironment to prevent melanoma progression[6], suggesting the pivotal role of Tet2 in regulating macrophage function in different context. In this study, we aim to dissect the molecular mechanism on how Tet2 deficiency disrupts normal macrophage function during heart repair.Methods: We crossed Tet2f/f mice with lysMCre mice to yield macrophage specific Tet2 deficient mice (Tet2f/f-lysMCre). We performed LAD ligation in Tet2f/f-lysMCre mice to induce MI. Age and gender matched lysMCre mice were used as controls. Cardiac function was monitored by echocardiogram every week after MI for 4 weeks. Then we sacrificed mice and performed histological analysis to evaluate heart damage and repairs in lysMCre and Tet2f/f-lysMCre mice. During the course of the experiments, we collected peripheral blood and bone marrows to examine the impact of Tet2 loss on macrophages in response to heart damage and repair. In parallel, we also collected heart tissue from lysMCre and Tet2f/f-lysMCre mice at 1 and 4 weeks after LAD ligation followed by single-cell RNA-seq analysis to further dissect the underlying molecular mechanism.Results: Prior to MI, no significant difference in heart function and macrophage numbers were observed between lysMCre and Tet2f/f-lysMCre mice at the age of 6-8 weeks old. Upon LAD-induced MI, we observed worse heart function in Tet2f/f-lysMCre mice compared to lysMCre mice. Tet2f/f-lysMCre mice showed ~40% reduction in ejection fraction and ~50% decrease in fraction shortening compared to lysMCre mice (n = 15). Further histological analysis revealed >2-fold increase in scar areas and infarcted areas in Tet2f/f-lysMCre compared to lysMCre mice. Single cell analysis confirmed a substantial increase of the fibroblast population in Tet2f/f-lysMCre mice upon LAD ligation. Furthermore, we also observed altered expression of a large fraction of genes related to macrophage activation and tissue repair capability, suggesting that Tet2 plays important roles in regulating macrophages function within damaged heart tissues. Further epigenomic analysis on sorted cardiac-specific macrophages is anticipated provide more valuable mechanistic information.Conclusion: Taken together, our data suggest that Tet2 deficient macrophages play an important role during heart damage and repair. Inactivation mutations of TET2 detected in CHIP patients might lead to impaired macrophage function during heart damage and therefore increase the CVD risk.

Therapeutic potential of regulatory macrophages generated from peritoneal dialysate in adriamycin nephropathy.

Cell therapy using macrophages requires large amounts of cells, which are difficult to collect from patients. Patients undergoing peritoneal dialysis (PD) discard huge numbers of peritoneal macrophages in dialysate daily. Macrophages can be modulated to become regulatory macrophages, which have shown great promise as a therapeutic strategy in experimental kidney disease and human kidney transplantation. This study aimed to examine the potential of using peritoneal macrophages (PMs) from peritoneal dialysate to treat kidney disease. Monocytes/macrophages accounted for >40% of total peritoneal leukocytes in both patients and mice undergoing PD. PMs from patients and mice undergoing PD were more mature than peripheral monocytes/macrophages, as shown by low expression of C-C motif chemokine receptor 2 (CCR2) and morphological changes during in vitro culture. PMs from patients and mice undergoing PD displayed normal macrophage function and could be modulated into a regulatory (M2) phenotype. In vivo, adoptive transfer of peritoneal M2 macrophages derived from PD mice effectively protected against kidney injury in mice with adriamycin nephropathy (AN). Importantly, the transfused peritoneal M2 macrophages maintained their M2 phenotype in kidney of AN mice. In conclusion, PMs derived from patients and mice undergoing PD exhibited conventional macrophage features. Peritoneal M2 macrophages derived from PD mice are able to reduce kidney injury in AN, suggesting that peritoneal macrophages from patients undergoing PD may have the potential for clinical therapeutic application.

Rho-specific Guanine nucleotide exchange factors (Rho-GEFs) inhibition affects macrophage phenotype and disrupts Golgi complex.

Macrophages play crucial role in tissue homeostasis and the innate and adaptive immune response. Depending on the state of activation macrophages acquire distinct phenotypes that depend on actin, which is regulated by small GTPase RhoA. The naive M0 macrophages are slightly elongated, pro-inflammatory M1 are round and M2 anti-inflammatory macrophages are elongated. We showed previously that interference with RhoA pathway (RhoA deletion or RhoA/ROCK kinase inhibition) disrupted actin, produced extremely elongated (hummingbird) macrophage phenotype and inhibited macrophage movement toward transplanted hearts. The RhoA function depends on the family of guanine-nucleotide exchange factors (GEFs), which catalyze the exchange of GDP for GTP and activate RhoA that reorganizes actin cytoskeleton. Using actin staining, immunostaining, Western blotting, flow cytometry and transmission electron microscopy we studied how a direct inhibition of Rho-GEFs with Rhosin (Rho GEF-binding domain blocker) and Y16 (Rho GEF DH-PH domain blocker) affects M0, M1 and M2 macrophage phenotypes. We also studied how Rho-GEFs inhibition and RhoA deletion affects organization of Golgi complex that is crucial for normal macrophage functions such as phagocytosis, antigen presentation and receptor recycling. We found that GEFs inhibition differently affected M0, M1 and M2 macrophages phenotype and that GEFs inhibition and RhoA deletion both caused changes in the ultrastructure of the Golgi complex. These results suggest that actin/RhoA- dependent shaping of macrophage phenotype has different requirements for activity of RhoA/GEFs pathway in M0, M1 and M2 macrophages, and that RhoA and Rho-GEFs functions are necessary for the maintenance of actin-dependent organization of Golgi complex.

Capsular specific IgM enhances complement-mediated phagocytosis and killing of Cryptococcus neoformans by methamphetamine-treated J774.16 macrophage-like cells

Methamphetamine (METH) is a powerful and highly addictive stimulant that affects the central nervous system of users in the United States and worldwide, and its consumption is associated to the acquisition of HIV and AIDS-related infections. METH enhances cryptococcosis in mice, an opportunistic infection caused by the encapsulated fungus Cryptococcus neoformans. Due to its ability to survive within macrophages, C. neoformans is an ideal model to study pathogen-macrophage interactions. METH abrogates normal macrophage function, which might contribute to the higher rate and more rapid progression of infections in drug abusers. Hence, we investigated the role of complement and specific IgM to C. neoformans capsular polysaccharide on the function of J774.16 macrophage-like cells after exposure to METH. We found that complement and IgM significantly promotes complement-mediated phagocytosis of C. neoformans by J774.16 cells in comparison to co-incubation with complement alone. IgM enhances the expression of complement receptor 3 on the surface macrophages treated with the drug. Also, IgM-increased macrophage phagocytosis of C. neoformans may be associated with upregulation of GTPase-RhoA, a key regulator of the actin polymerization signaling cascade. Fungal cells incubated with complement and IgM in the presence of METH demonstrated higher number of cells per aggregate, a possible explanation for their enhanced ingestion by phagocytes. IgM increased killing of yeast cells by macrophages by inhibiting the alkalization of the phagosome and stimulating the intracellular production of nitric oxide. Together, our findings suggest that IgM stimulates the effector functions of macrophages against opportunistic pathogens in the setting of drug abuse.

New Macrophage Models of Gaucher Disease Offer New Tools for Drug Development

Gaucher disease is an inherited enzyme deficiency resulting in the lysosomal accumulation of specific glycolipids in macrophages and, in some cases, neurons. While current treatments are effective at reducing this glycolipid storage in macrophages, they are expensive and ineffective in treating neurological manifestations of the disease, driving the search for novel therapeutics. Moreover, mutations in GBA1, the gene implicated in Gaucher disease, are an important risk factor for the development of Parkinson disease and related disorders, an association that has further heightened interest in Gaucher disease research. However, the development of therapeutic strategies has been hampered by a shortage of appropriate cellular models of Gaucher disease. We have generated two novel macrophage models of Gaucher disease, one through the differentiation of peripheral blood monocytes from patients with Gaucher disease and the other through the differentiation of induced pluripotent stem cells derived from patient fibroblasts. Both disease models demonstrate similar cellular phenotypes and exhibit extensive glycolipid storage when exposed to exogenous lipid sources such as erythrocyte membranes. Furthermore, we have used these models to confirm the efficacy of a novel small molecule in clearing glycolipid storage and restoring normal macrophage function. These results demonstrate the usefulness of these models in exploring new therapeutics for Gaucher disease and related disorders.

Pikfyve Is Essential for Lysosomal Homeostasis in Macrophages

The biogenesis and degradative functions of the lysosome in macrophages and other cells is critically dependent on the membrane trafficking along the endolysosomal system. This trafficking is tightly regulated by several signaling molecules including the endosomal phosphoinositide PtdIns(3,5)P2, which is synthesized from PtdIns(3)P in mammals by the lipid kinase, PIKfyve. Although PtdIns(3,5)P2 deficiency is associated with neurodegeneration in mice and humans, the critical importance of PIKfyve to normal macrophage function is poorly understood. To address this, we genetically engineered mice to lack PIKfyve in their macrophages by pairing PIKfyvefl/fl mice with mice that are transgenic for the myeloid-specific LysM-Cre (PIKfyvefl/fl LysM-Cre). PIKfyvefl/fl LysM-Cre mice develop weight gain and massive hepatosplenomegaly. Immunohistochemical analyses of the liver and spleen from PIKfyvefl/fl LysM-Cre mice demonstrate tissue infiltration of mononuclear cells with numerous cytoplasmic vacuoles that stain with the macrophage-specific marker, anti-F4/80 antibody. To verify that these vacuolated cells result from PIKfyve ablation, we utilized genetic lineage tracing by crossing PIKfyvefl/fl LysM-Cre mice with mice transgenic for a Cre-dependent β-galactosidase reporter. Histochemical analyses using a chromogenic substrate, x-gal, reveal β-galactosidase activity in cells with small cytoplasmic vacuoles, demonstrating Cre expression and therefore PIKfyve ablation in these cells. However, β-galactosidase activity is absent in cells with large vacuoles, indicating that these cells do not express Cre and still contain PIKfyve. These findings suggest that the cytoplasmic vacuolation resulted from both cell-autonomous and non-cell-autonomous effects of PIKfyve deletion. To investigate the macrophage-specific effects of PIKfyve ablation in the lysosomal biogenesis and functions, we isolated macrophages from the bone marrow and spleen of PIKfyvefl/fl LysM-Cre mice using magnetic beads bound to anti-F4/80 antibody. Immunoblot analysis of F4/80+ PIKfyve-null macrophages reveal increased expression of lysosomal proteins including Cathepsin D, Cathepsin S, and LAMP-1, demonstrating that PIKfyve is essential for lysosome homeostasis in macrophages.Recent studies showed that the transcription factor TFEB is a master regulator of lysosomal biogenesis and function. When TFEB is phosphorylated by mTORC1 on the lysosomal membrane, TFEB is retained in the cytosol, and therefore unable to promote gene transcription. However, when TFEB is de-phosphorylated, TFEB translocates to the nucleus where it is transcriptionally active. Since PtdIns(3,5)P2 was previously shown to regulate the recruitment of mTORC1 to the lysosome, we hypothesized that the synthesis of PtdIns(3,5)P2 by PIKfyve regulates TFEB phosphorylation and indirectly controls TFEB transcriptional activity. To test this hypothesis, we studied the expression of TFEB in the F4/80+ macrophages isolated from the control and PIKfyvefl/fl LysM-Cre mice. While wild-type macrophages express TFEB in both phosphorylated and dephosphorylated forms, PIKfyve-null macrophages primarily express the dephosphorylated form of TFEB. This suggests that PIKfyve is required for the phosphorylation of TFEB and consequently PIKfyve prevents TFEB’s ability to transcribe lysosomal genes. Thus, PIKfyve ablation within macrophages upregulates the transcription of lysosomal genes via TFEB activation. Our study suggests that PIKfyve is a critical regulator of lysosomal homeostasis and demonstrates a previously unrecognized link between PIKfyve, PtdIns(3,5)P2 synthesis, and the TFEB pathway in the regulation of lysosomal biogenesis and function within macrophages. DisclosuresNo relevant conflicts of interest to declare.

Analysis of the misregulation of RhoA and RhoH in autoimmune mice. (HUM7P.302)

Abstract When cultured in the presence of apoptotic cells, cell bodies or lipids, RhoA is misregulated in macrophages from all 6 autoimmune mouse strains but none of the 7 normal strains tested. Misregulation of RhoA results in defective morphology, altered cytokine production, and increased adhesion. The same macrophages cultured without apoptotic cell lipids had properly regulated RhoA and behaved normally. Normal mouse macrophages cultured with the Rho inhibitor C3 toxin, had defective macrophage morphology and behavior. Therefore, we proposed that autoimmunity is due to an apoptotic cell recognition signaling defect and that normal macrophage function may be restored by a chemical reversal of the defect. Our new data demonstrate that lupus strain, MRL, macrophages cultured in the presence of C3 no longer had defects in morphology and function. The fact that a RhoA inhibitor restored RhoA function indicates that the misregulation in autoimmune macrophages involves a RhoA regulator also affected by C3 and not RhoA itself. RhoH is a known inhibitor of RhoA and it is only expressed in hematopoietic cells. Experiments comparing RhoH expression in BALB and MRL macrophages suggest at least four different sizes of RhoH protein are expressed in normal macrophages in response to lipid exposure and that lupus macrophages do not express the multiple forms of RhoH even in the presence of lipids. A lack of proper RhoH expression could be behind the autoimmune macrophage defect.

Transient Receptor Potential Ion Channels

This article reviews the current understanding of transient receptor potential ion channels (TRP channels) in health and disease. Transient receptor potential ion channels are a group of 27 channels that are expressed in all tissues. These channels play important roles in surgically important problems, such as chronic pain, susceptibility to infection, hypothermia, and some cancers. A literature search was performed. This review focuses on the role of TRP channels in a few surgically important disease processes, such as pain, inflammation, airway diseases, and malignant melanomas. In addition, we discuss some of the structural properties that are important for the activation of TRP channels. TRPA1 and TRPV1 are expressed on pain fibers and play an important role in the development of chronic pain, such as chemotherapy-related neuropathic pain. Deletion of TRPA1 and TRPV1 suppresses the development of chronic pain, and blockers of TRPA1 and TRPV1 show promise as a new class of painkillers. Furthermore, several TRP channels are expressed on immune cells. Macrophages express at least 3 different TRP channels, and the properly balanced activation of all these channels together allows normal macrophage function. Deletion of any of these channels results in impaired macrophage function and increased susceptibility to infection. Because several of these TRP channels on macrophages are temperature sensitive, they may comprise the link for hypothermia-related infectious complications in trauma, and to a lesser degree, in elective surgical patients. Transient receptor potential ion channels are involved in several surgically important disease processes. Activation or blockade of these channels offers new therapeutic opportunities. Pharmacologic activation or blockade of TRP channels may offer new treatment options in surgical patients for the management of pain and infections.

Innate Immune Response to Streptococcus iniae Infection in Zebrafish Larvae

Streptococcus iniae causes systemic infection characterized by meningitis and sepsis. Here, we report a larval zebrafish model of S. iniae infection. Injection of wild-type S. iniae into the otic vesicle induced a lethal infection by 24 h postinfection. In contrast, an S. iniae mutant deficient in polysaccharide capsule (cpsA mutant) was not lethal, with greater than 90% survival at 24 h postinfection. Live imaging demonstrated that both neutrophils and macrophages were recruited to localized otic infection with mutant and wild-type S. iniae and were able to phagocytose bacteria. Depletion of neutrophils and macrophages impaired host survival following infection with wild-type S. iniae and the cpsA mutant, suggesting that leukocytes are critical for host survival in the presence of both the wild-type and mutant bacteria. However, zebrafish larvae with impaired neutrophil function but normal macrophage function had increased susceptibility to wild-type bacteria but not the cpsA mutant. Taking these findings together, we have developed a larval zebrafish model of S. iniae infection and have found that although neutrophils are important for controlling infection with wild-type S. iniae, neutrophils are not necessary for host defense against the cpsA mutant.

Cytokine production in immortalized porcine alveolar macrophages infected with porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically significant viral pathogens for pig production worldwide. PRRSV primarily infects cells of the monocyte/macrophage lineage such as porcine alveolar macrophages (PAMs), and is generally known to suppress normal macrophage function and regulate innate immune responses to viral infection. A continuous PRRSV-permissive porcine monocyte-derived cell line was previously generated to facilitate virus propagation and advance research on the biology and immunology of PRRSV. With the availability of this valuable tool, we first sought to explore modulation of inflammatory cytokine expression in PAM-pCD163 cells infected with each genotype PRRSV and to establish an in vitro system for immune function studies using PRRSV isolates.

Human Amnion Epithelial Cells do Not Abrogate Pulmonary Fibrosis in Mice with Impaired Macrophage Function

Since current treatments for both acute and chronic lung diseases are less than ideal, there has been recent interest in the use of cell-based therapies for inflammatory lung disease. Specifically, human amnion epithelial cells (hAECs) have been shown to reduce bleomycin-induced lung injury and prevent subsequent loss of respiratory function, primarily through modulation of the host immune response. The precise mechanisms of this effect remain unclear. We aimed to investigate the potential of hAECs to mitigate bleomycin-induced lung injury in surfactant protein C deficient (Sftpc(−/−)) mice, which are highly susceptible to pulmonary injury as a result of impairment of macrophage function. Primary hAECs were administered to wild-type (Sftpc(+/+)) and Sftpc(−/−) mice 24 h after exposure to bleomycin. Compared to Sftpc(+/+) mice receiving bleomycin alone, Sftpc(+/+) mice administered hAECs 24 h after bleomycin exposure had decreased expression of proinflammatory genes, decreased macrophage and neutrophil infiltration, fibrosis, collagen content, and α-smooth muscle actin as well as a significant improvement in lung function. Compared to Sftpc(−/−) mice given bleomycin alone, Sftpc(−/−) mice administered hAECs 24 h after bleomycin did not have a decrease in inflammatory gene expression or a reduction in macrophage pulmonary infiltration. Subsequently, Sftpc(−/−) mice did not show any decrease in pulmonary fibrosis or improvement of lung function after hAEC administration. The ability of hAECs to mitigate bleomycin-induced lung injury is abolished in Sftpc(−/−) mice, suggesting that hAECs require normal host macrophage function to exert their reparative effects.

WS17.3 Alteration of human macrophage functions in cystic fibrosis

Recently, several reports suggest that CFTR should contribute to altered inflam-matory response in cystic fibrosis (CF) by modification of normal macrophagefunctions. In order to highlight possible intrinsic macrophage defects due to CFTRdysfunction, we have studied macrophages from human peripheral blood monocytesof healthy subjects (non CF) and CF patients.Ficoll isolated adherent monocytes were differentiated in macrophages with GM-CSF (400U/mL) during 6 days. Non CF macrophages were treated during 72h withCFTRinh−172(10, 20mM). We have focused to study some membrane markers ofCD71+macrophages (mCD14, CD16, CD64, CD11b) by flow cytometry. SolubleCD14, IL-1b and IL-8 supernatant levels were quantified by ELISA. Phagocytosiswas measured by fluorescein-labeledE. coliBioparticles.In CF macrophages, we have observed strong inhibition of phagocytosis (70%)as well as 1.3-, 1.8- and 2.0-fold significant decrease of CD16, CD64 andCD11b respectively. However membrane CD14 (mCD14) was slightly increased butmCD14/sCD14 ratio is in favor of sCD14 (1/6vs1/270 in CF cells). Elsewhere,concentrations of IL-1b and IL-8 were strongly raised in CF cell supernatant. CFTRinhibition in non CF macrophages also showed alterations in surface expression ofCD11b, CD16, CD64, IL-8 level and phagocytosis.Pathogens recognition and phagocytosis seems to be impaired in CF macrophages,and some alterations could be associated with CFTR defect. Thus, perturbationsof CD11b and sCD14, involved in innate immune response, may play a potentialimportant role in CF pathogenesis. [ No other pdf]