Mucus sialylation determines intestinal host-commensal homeostasis.

Intestinal mucus forms the first line of defense against bacterial invasion while providing nutrition to support microbial symbiosis. How the host controls mucus barrier integrity and commensalism is unclear. We show that terminal sialylation of glycans on intestinal mucus by ST6GALNAC1 (ST6), the dominant sialyltransferase specifically expressed in goblet cells and induced by microbial pathogen-associated molecular patterns, is essential for mucus integrity and protecting against excessive bacterial proteolytic degradation. Glycoproteomic profiling and biochemical analysis of ST6 mutations identified in patients show that decreased sialylation causes defective mucus proteins and congenital inflammatory bowel disease (IBD). Mice harboring a patient ST6 mutation have compromised mucus barriers, dysbiosis, and susceptibility to intestinal inflammation. Based on our understanding of the ST6 regulatory network, we show that treatment with sialylated mucin or a Foxo3 inhibitor can ameliorate IBD.

GIMAP5 deficiency reveals a mammalian ceramide-driven longevity assurance pathway.

Preserving cells in a functional, non-senescent state is a major goal for extending human healthspans. Model organisms reveal that longevity and senescence are genetically controlled, but how genes control longevity in different mammalian tissues is unknown. Here, we report a new human genetic disease that causes cell senescence, liver and immune dysfunction, and early mortality that results from deficiency of GIMAP5, an evolutionarily conserved GTPase selectively expressed in lymphocytes and endothelial cells. We show that GIMAP5 restricts the pathological accumulation of long-chain ceramides (CERs), thereby regulating longevity. GIMAP5 controls CER abundance by interacting with protein kinase CK2 (CK2), attenuating its ability to activate CER synthases. Inhibition of CK2 and CER synthase rescues GIMAP5-deficient T cells by preventing CER overaccumulation and cell deterioration. Thus, GIMAP5 controls longevity assurance pathways crucial for immune function and healthspan in mammals.

Congenital iRHOM2 deficiency causes ADAM17 dysfunction and environmentally directed immunodysregulatory disease.

We report a pleiotropic disease due to loss-of-function mutations in RHBDF2, the gene encoding iRHOM2, in two kindreds with recurrent infections in different organs. One patient had recurrent pneumonia but no colon involvement, another had recurrent infectious hemorrhagic colitis but no lung involvement and the other two experienced recurrent respiratory infections. Loss of iRHOM2, a rhomboid superfamily member that regulates the ADAM17 metalloproteinase, caused defective ADAM17-dependent cleavage and release of cytokines, including tumor-necrosis factor and amphiregulin. To understand the diverse clinical phenotypes, we challenged Rhbdf2-/- mice with Pseudomonas aeruginosa by nasal gavage and observed more severe pneumonia, whereas infection with Citrobacter rodentium caused worse inflammatory colitis than in wild-type mice. The fecal microbiota in the colitis patient had characteristic oral species that can predispose to colitis. Thus, a human immunodeficiency arising from iRHOM2 deficiency causes divergent disease phenotypes that can involve the local microbial environment.

NAD activates olfactory receptor 1386 to regulate type I interferon responses in Plasmodium yoelii YM infection.

Olfactory receptors (Olfr) are G protein-coupled receptors that are normally expressed on olfactory sensory neurons to detect volatile chemicals or odorants. Interestingly, many Olfrs are also expressed in diverse tissues and function in cell-cell recognition, migration, and proliferation as well as immune responses and disease processes. Here, we showed that many Olfr genes were expressed in the mouse spleen, linked to Plasmodium yoelii genetic loci significantly, and/or had genome-wide patterns of LOD scores (GPLSs) similar to those of host Toll-like receptor genes. Expression of specific Olfr genes such as Olfr1386 in HEK293T cells significantly increased luciferase signals driven by IFN-ß and NF-κB promoters, with elevated levels of phosphorylated TBK1, IRF3, P38, and JNK. Mice without Olfr1386 were generated using the CRISPR/Cas9 method, and the Olfr1386-/- mice showed significantly lower IFN-α/ß levels and longer survival than wild-type (WT) littermates after infection with P. yoelii YM parasites. Inhibition of G protein signaling and P38 activity could affect cyclic AMP-responsive element promoter-driven luciferase signals and IFN-ß mRNA levels in HEK293T cells expressing the Olfr1386 gene, respectively. Screening of malaria parasite metabolites identified nicotinamide adenine dinucleotide (NAD) as a potential ligand for Olfr1386, and NAD could stimulate IFN-ß responses and phosphorylation of TBK1 and STAT1/2 in RAW264.7 cells. Additionally, parasite RNA (pRNA) could significantly increase Olfr1386 mRNA levels. This study links multiple Olfrs to host immune response pathways, identifies a candidate ligand for Olfr1386, and demonstrates the important roles of Olfr1386 in regulating type I interferon (IFN-I) responses during malaria parasite infections.

Enhancement of LIN28B-induced hematopoietic reprogramming by IGF2BP3.

Fetal hematopoietic stem and progenitor cells (HSPCs) hold promise to cure a wide array of hematological diseases, and we previously found a role for the RNA-binding protein (RBP) Lin28b in respecifying adult HSPCs to resemble their fetal counterparts. Here we show by single-cell RNA sequencing that Lin28b alone was insufficient for complete reprogramming of gene expression from the adult toward the fetal pattern. Using proteomics and in situ analyses, we found that Lin28b (and its closely related paralog, Lin28a) directly interacted with Igf2bp3, another RBP, and their enforced co-expression in adult HSPCs reactivated fetal-like B-cell development in vivo more efficiently than either factor alone. In B-cell progenitors, Lin28b and Igf2bp3 jointly stabilized thousands of mRNAs by binding at the same sites, including those of the B-cell regulators Pax5 and Arid3a as well as Igf2bp3 mRNA itself, forming an autoregulatory loop. Our results suggest that Lin28b and Igf2bp3 are at the center of a gene regulatory network that mediates the fetal-adult hematopoietic switch. A method to efficiently generate induced fetal-like hematopoietic stem cells (ifHSCs) will facilitate basic studies of their biology and possibly pave a path toward their clinical application.

Clinical and functional spectrum of RAC2-related immunodeficiency.

ABSTRACT: Mutations in the small Rho-family guanosine triphosphate hydrolase RAC2, critical for actin cytoskeleton remodeling and intracellular signal transduction, are associated with neonatal severe combined immunodeficiency (SCID), infantile neutrophilic disorder resembling leukocyte adhesion deficiency (LAD), and later-onset combined immune deficiency (CID). We investigated 54 patients (23 previously reported) from 37 families yielding 15 novel RAC2 missense mutations, including one present only in homozygosity. Data were collected from referring physicians and literature reports with updated clinical information. Patients were grouped by presentation: neonatal SCID (n = 5), infantile LAD-like disease (n = 5), or CID (n = 44). Disease correlated to RAC2 activity: constitutively active RAS-like mutations caused neonatal SCID, dominant-negative mutations caused LAD-like disease, whereas dominant-activating mutations caused CID. Significant T- and B-lymphopenia with low immunoglobulins were seen in most patients; myeloid abnormalities included neutropenia, altered oxidative burst, impaired neutrophil migration, and visible neutrophil macropinosomes. Among 42 patients with CID with clinical data, upper and lower respiratory infections and viral infections were common. Twenty-three distinct RAC2 mutations, including 15 novel variants, were identified. Using heterologous expression systems, we assessed downstream effector functions including superoxide production, p21-activated kinase 1 binding, AKT activation, and protein stability. Confocal microscopy showed altered actin assembly evidenced by membrane ruffling and macropinosomes. Altered protein localization and aggregation were observed. All tested RAC2 mutant proteins exhibited aberrant function; no single assay was sufficient to determine functional consequence. Most mutants produced elevated superoxide; mutations unable to support superoxide formation were associated with bacterial infections. RAC2 mutations cause a spectrum of immune dysfunction, ranging from early onset SCID to later-onset combined immunodeficiencies depending on RAC2 activity. This trial was registered at www.clinicaltrials.gov as #NCT00001355 and #NCT00001467.

The V2 domain of HIV gp120 mimics an interaction between CD4 and integrin ⍺4ß7.

The CD4 receptor, by stabilizing TCR-MHC II interactions, plays a central role in adaptive immunity. It also serves as the HIV docking receptor. The HIV gp120 envelope protein binds directly to CD4. This interaction is a prerequisite for viral entry. gp120 also binds to ⍺4ß7, an integrin that is expressed on a subset of memory CD4+ T cells. HIV tropisms for CD4+ T cells and gut tissues are central features of HIV pathogenesis. We report that CD4 binds directly to ⍺4ß7 in a dynamic way, consistent with a cis regulatory interaction. The molecular details of this interaction are related to the way in which gp120 interacts with both receptors. Like MAdCAM-1 and VCAM-1, two recognized ligands of ⍺4ß7, the binding interface on CD4 includes 2 sites (1° and accessory), distributed across its two N-terminal IgSF domains (D1 and D2). The 1° site includes a sequence in the G ß-strand of CD4 D2, KIDIV, that binds directly to ⍺4ß7. This pentapeptide sequence occurs infrequently in eukaryotic proteins. However, a closely related and conserved sequence, KLDIV, appears in the V2 domain of gp120. KLDIV mediates gp120-⍺4ß7 binding. The accessory ⍺4ß7 binding site on CD4 includes Phe43. The Phe43 aromatic ring protrudes outward from one edge of a loop connecting the C'C" strands of CD4 D1. Phe43 is a principal contact for HIV gp120. It interacts with conserved residues in the recessed CD4 binding pocket. Substitution of Phe43 abrogates CD4 binding to both gp120 and ⍺4ß7. As such, the interactions of gp120 with both CD4 and ⍺4ß7 reflect elements of their interactions with each other. These findings indicate that gp120 specificities for CD4 and ⍺4ß7 are interrelated and suggest that selective pressures which produced a CD4 tropic virus that replicates in gut tissues are linked to a dynamic interaction between these two receptors.

A processing product of the Plasmodium falciparum reticulocyte binding protein RH1 shows a close association with AMA1 during junction formation.

Plasmodium falciparum responsible for the most virulent form of malaria invades human erythrocytes through multiple ligand-receptor interactions. The P. falciparum reticulocyte binding protein homologues (PfRHs) are expressed at the apical end of merozoites and form interactions with distinct erythrocyte surface receptors that are important for invasion. Here using a range of monoclonal antibodies (mAbs) against different regions of PfRH1 we have investigated the role of PfRH processing during merozoite invasion. We show that PfRH1 gets differentially processed during merozoite maturation and invasion and provide evidence that the different PfRH1 processing products have distinct functions during invasion. Using in-situ Proximity Ligation and FRET assays that allow probing of interactions at the nanometre level we show that a subset of PfRH1 products form close association with micronemal proteins Apical Membrane Antigen 1 (AMA1) in the moving junction suggesting a critical role in facilitating junction formation and active invasion. Our data provides evidence that time dependent processing of PfRH proteins is a mechanism by which the parasite is able to regulate distinct functional activities of these large processes. The identification of a specific close association with AMA1 in the junction now may also provide new avenues to target these interactions to prevent merozoite invasion.

Aedes aegypti Piwi4 Structural Features Are Necessary for RNA Binding and Nuclear Localization.

The PIWI-interacting RNA (piRNA) pathway provides an RNA interference (RNAi) mechanism known from Drosophila studies to maintain the integrity of the germline genome by silencing transposable elements (TE). Aedes aegypti mosquitoes, which are the key vectors of several arthropod-borne viruses, exhibit an expanded repertoire of Piwi proteins involved in the piRNA pathway, suggesting functional divergence. Here, we investigate RNA-binding dynamics and subcellular localization of A. aegypti Piwi4 (AePiwi4), a Piwi protein involved in antiviral immunity and embryonic development, to better understand its function. We found that AePiwi4 PAZ (Piwi/Argonaute/Zwille), the domain that binds the 3' ends of piRNAs, bound to mature (3' 2' O-methylated) and unmethylated RNAs with similar micromolar affinities (KD = 1.7 ± 0.8 µM and KD of 5.0 ± 2.2 µM, respectively; p = 0.05) in a sequence independent manner. Through site-directed mutagenesis studies, we identified highly conserved residues involved in RNA binding and found that subtle changes in the amino acids flanking the binding pocket across PAZ proteins have significant impacts on binding behaviors, likely by impacting the protein secondary structure. We also analyzed AePiwi4 subcellular localization in mosquito tissues. We found that the protein is both cytoplasmic and nuclear, and we identified an AePiwi4 nuclear localization signal (NLS) in the N-terminal region of the protein. Taken together, these studies provide insights on the dynamic role of AePiwi4 in RNAi and pave the way for future studies aimed at understanding Piwi interactions with diverse RNA populations.

Identification of Poxvirus Genome Uncoating and DNA Replication Factors with Mutually Redundant Roles.

Genome uncoating is essential for replication of most viruses. For poxviruses, the process is divided into two stages: removal of the envelope, allowing early gene expression, and breaching of the core wall, allowing DNA release, replication, and late gene expression. Subsequent studies showed that the host proteasome and the viral D5 protein, which has an essential role in DNA replication, are required for vaccinia virus (VACV) genome uncoating. In a search for additional VACV uncoating proteins, we noted a report that described a defect in DNA replication and late expression when the gene encoding a 68-kDa ankyrin repeat/F-box protein (68k-ank), associated with the cellular SCF (Skp1, cullin1, F-box-containing complex) ubiquitin ligase complex, was deleted from the attenuated modified vaccinia virus Ankara (MVA). Here we showed that the 68k-ank deletion mutant exhibited diminished genome uncoating, formation of DNA prereplication sites, and degradation of viral cores as well as an additional, independent defect in DNA synthesis. Deletion of the 68k-ank homolog of VACV strain WR, however, was without effect, suggesting the existence of compensating genes. By inserting VACV genes into an MVA 68k-ank deletion mutant, we discovered that M2, a member of the poxvirus immune evasion (PIE) domain superfamily and a regulator of NF-κB, and C5, a member of the BTB/Kelch superfamily associated with cullin-3-based ligase complexes, independently rescued the 68k-ank deletion phenotype. Thus, poxvirus uncoating and DNA replication are intertwined processes involving at least three viral proteins with mutually redundant functions in addition to D5.IMPORTANCE Poxviruses comprise a family of large DNA viruses that infect vertebrates and invertebrates and cause diseases of medical and zoological importance. Poxviruses, unlike most other DNA viruses, replicate in the cytoplasm, and their large genomes usually encode 200 or more proteins with diverse functions. About 90 genes may be essential for chordopoxvirus replication based either on their conservation or individual gene deletion studies. However, this number may underestimate the true number of essential functions because of redundancy. Here we show that any one of three seemingly unrelated and individually nonessential proteins is required for the incompletely understood processes of genome uncoating and DNA replication, an example of synthetic lethality. Thus, poxviruses appear to have a complex genetic interaction network that has not been fully appreciated and which will require multifactor deletion screens to assess.

Influenza NS1 directly modulates Hedgehog signaling during infection.

The multifunctional NS1 protein of influenza A viruses suppresses host cellular defense mechanisms and subverts other cellular functions. We report here on a new role for NS1 in modifying cell-cell signaling via the Hedgehog (Hh) pathway. Genetic epistasis experiments and FRET-FLIM assays in Drosophila suggest that NS1 interacts directly with the transcriptional mediator, Ci/Gli1. We further confirmed that Hh target genes are activated cell-autonomously in transfected human lung epithelial cells expressing NS1, and in infected mouse lungs. We identified a point mutation in NS1, A122V, that modulates this activity in a context-dependent fashion. When the A122V mutation was incorporated into a mouse-adapted influenza A virus, it cell-autonomously enhanced expression of some Hh targets in the mouse lung, including IL6, and hastened lethality. These results indicate that, in addition to its multiple intracellular functions, NS1 also modifies a highly conserved signaling pathway, at least in part via cell autonomous activities. We discuss how this new Hh modulating function of NS1 may influence host lethality, possibly through controlling cytokine production, and how these new insights provide potential strategies for combating infection.

IL-20 Signaling in Activated Human Neutrophils Inhibits Neutrophil Migration and Function.

Neutrophils possess multiple antimicrobial mechanisms that are critical for protection of the host against infection with extracellular microbes, such as the bacterial pathogen Staphylococcus aureus Recruitment and activation of neutrophils at sites of infection are driven by cytokine and chemokine signals that directly target neutrophils via specific cell surface receptors. The IL-20 subfamily of cytokines has been reported to act at epithelial sites and contribute to psoriasis, wound healing, and anti-inflammatory effects during S. aureus infection. However, the ability of these cytokines to directly affect neutrophil function remains incompletely understood. In this article, we show that human neutrophils altered their expression of IL-20R chains upon migration and activation in vivo and in vitro. Such activation of neutrophils under conditions mimicking infection with S. aureus conferred responsiveness to IL-20 that manifested as modification of actin polymerization and inhibition of a broad range of actin-dependent functions, including phagocytosis, granule exocytosis, and migration. Consistent with the previously described homeostatic and anti-inflammatory properties of IL-20 on epithelial cells, the current study provides evidence that IL-20 directly targets and inhibits key inflammatory functions of neutrophils during infection with S. aureus.

IRAV (FLJ11286), an Interferon-Stimulated Gene with Antiviral Activity against Dengue Virus, Interacts with MOV10.

Dengue virus (DENV) is a member of the genus Flavivirus and can cause severe febrile illness. Here, we show that FLJ11286, which we refer to as IRAV, is induced by DENV in an interferon-dependent manner, displays antiviral activity against DENV, and localizes to the DENV replication complex. IRAV is an RNA binding protein and localizes to cytoplasmic processing bodies (P bodies) in uninfected cells, where it interacts with the MOV10 RISC complex RNA helicase, suggesting a role for IRAV in the processing of viral RNA. After DENV infection, IRAV, along with MOV10 and Xrn1, localizes to the DENV replication complex and associates with DENV proteins. Depletion of IRAV or MOV10 results in an increase in viral RNA. These data serve to characterize an interferon-stimulated gene with antiviral activity against DENV, as well as to propose a mechanism of activity involving the processing of viral RNA. IMPORTANCE Dengue virus, a member of the family Flaviviridae, can result in a life-threatening illness and has a significant impact on global health. Dengue virus has been shown to be particularly sensitive to the effects of type I interferon; however, little is known about the mechanisms by which interferon-stimulated genes function to inhibit viral replication. A better understanding of the interferon-mediated antiviral response to dengue virus may aid in the development of novel therapeutics. Here, we examine the influence of the interferon-stimulated gene IRAV (FLJ11286) on dengue virus replication. We show that IRAV associates with P bodies in uninfected cells and with the dengue virus replication complex after infection. IRAV also interacts with MOV10, depletion of which is associated with increased viral replication. Our results provide insight into a newly identified antiviral gene, as well as broadening our understanding of the innate immune response to dengue virus infection.

Actin-Related Protein 2 (ARP2) and Virus-Induced Filopodia Facilitate Human Respiratory Syncytial Virus Spread.

Human respiratory syncytial virus (RSV) is an enveloped RNA virus that is the most important viral cause of acute pediatric lower respiratory tract illness worldwide, and lacks a vaccine or effective antiviral drug. The involvement of host factors in the RSV replicative cycle remains poorly characterized. A genome-wide siRNA screen in human lung epithelial A549 cells identified actin-related protein 2 (ARP2) as a host factor involved in RSV infection. ARP2 knockdown did not reduce RSV entry, and did not markedly reduce gene expression during the first 24 hr of infection, but decreased viral gene expression thereafter, an effect that appeared to be due to inhibition of viral spread to neighboring cells. Consistent with reduced spread, there was a 10-fold reduction in the release of infectious progeny virions in ARP2-depleted cells at 72 hr post-infection. In addition, we found that RSV infection induced filopodia formation and increased cell motility in A549 cells and that this phenotype was ARP2 dependent. Filopodia appeared to shuttle RSV to nearby uninfected cells, facilitating virus spread. Expression of the RSV F protein alone from a plasmid or heterologous viral vector in A549 cells induced filopodia, indicating a new role for the RSV F protein, driving filopodia induction and virus spread. Thus, this study identified roles for ARP2 and filopodia in RSV-induced cell motility, RSV production, and RSV cell-to-cell spread.

A single nucleotide polymorphism in the Plasmodium falciparum atg18 gene associates with artemisinin resistance and confers enhanced parasite survival under nutrient deprivation.

BACKGROUND: Artemisinin-resistant Plasmodium falciparum has been reported throughout the Greater Mekong subregion and threatens to disrupt current malaria control efforts worldwide. Polymorphisms in kelch13 have been associated with clinical and in vitro resistance phenotypes; however, several studies suggest that the genetic determinants of resistance may involve multiple genes. Current proposed mechanisms of resistance conferred by polymorphisms in kelch13 hint at a connection to an autophagy-like pathway in P. falciparum. RESULTS: A SNP in autophagy-related gene 18 (atg18) was associated with long parasite clearance half-life in patients following artemisinin-based combination therapy. This gene encodes PfAtg18, which is shown to be similar to the mammalian/yeast homologue WIPI/Atg18 in terms of structure, binding abilities, and ability to form puncta in response to stress. To investigate the contribution of this polymorphism, the atg18 gene was edited using CRISPR/Cas9 to introduce a T38I mutation into a k13-edited Dd2 parasite. The presence of this SNP confers a fitness advantage by enabling parasites to grow faster in nutrient-limited settings. The mutant and parent parasites were screened against drug libraries of 6349 unique compounds. While the SNP did not modulate the parasite's susceptibility to any of the anti-malarial compounds using a 72-h drug pulse, it did alter the parasite's susceptibility to 227 other compounds. CONCLUSIONS: These results suggest that the atg18 T38I polymorphism may provide additional resistance against artemisinin derivatives, but not partner drugs, even in the absence of kelch13 mutations, and may also be important in parasite survival during nutrient deprivation.

Live cell imaging unveils multiple domain requirements for in vivo dimerization of the glucocorticoid receptor.

Glucocorticoids are essential for life, but are also implicated in disease pathogenesis and may produce unwanted effects when given in high doses. Glucocorticoid receptor (GR) transcriptional activity and clinical outcome have been linked to its oligomerization state. Although a point mutation within the GR DNA-binding domain (GRdim mutant) has been reported as crucial for receptor dimerization and DNA binding, this assumption has recently been challenged. Here we have analyzed the GR oligomerization state in vivo using the number and brightness assay. Our results suggest a complete, reversible, and DNA-independent ligand-induced model for GR dimerization. We demonstrate that the GRdim forms dimers in vivo whereas adding another mutation in the ligand-binding domain (I634A) severely compromises homodimer formation. Contrary to dogma, no correlation between the GR monomeric/dimeric state and transcriptional activity was observed. Finally, the state of dimerization affected DNA binding only to a subset of GR binding sites. These results have major implications on future searches for therapeutic glucocorticoids with reduced side effects.

Microfilariae of Brugia malayi Inhibit the mTOR Pathway and Induce Autophagy in Human Dendritic Cells.

Immune modulation is a hallmark of patent filarial infection, including suppression of antigen-presenting cell function and downmodulation of filarial antigen-specific T cell responses. The mammalian target of rapamycin (mTOR) signaling pathway has been implicated in immune regulation, not only by suppressing T cell responses but also by regulating autophagy (through mTOR sensing amino acid availability). Global proteomic analysis (liquid chromatography-tandem mass spectrometry) of microfilaria (mf)-exposed monocyte-derived dendritic cells (DC) indicated that multiple components of the mTOR signaling pathway, including mTOR, eIF4A, and eIF4E, are downregulated by mf, suggesting that mf target this pathway for immune modulation in DC. Utilizing Western blot analysis, we demonstrate that similar to rapamycin (a known mTOR inhibitor), mf downregulate the phosphorylation of mTOR and its regulatory proteins, p70S6K1 and 4E-BP1, a process essential for DC protein synthesis. As active mTOR signaling regulates autophagy, we examined whether mf exposure alters autophagy-associated processes. mf-induced autophagy was reflected in marked upregulation of phosphorylated Beclin 1, known to play an important role in both autophagosome formation and autolysosome fusion, in induction of LC3II, a marker of autophagosome formation, and in induced degradation of p62, a ubiquitin-binding protein that aggregates protein in autophagosomes and is degraded upon autophagy that was reduced significantly by mf exposure and by rapamycin. Together, these results suggest that Brugia malayi mf employ mechanisms of metabolic modulation in DC to influence the regulation of the host immune response by downregulating mTOR signaling, resulting in increased autophagy. Whether this is a result of the parasite-secreted rapamycin homolog is currently under study.

CXCR4 antagonist AMD3100 redistributes leukocytes from primary immune organs to secondary immune organs, lung, and blood in mice.

AMD3100 (plerixafor), is a specific CXCR4 antagonist approved by the FDA for mobilizing hematopoietic stem cells from bone marrow to blood for transplantation in cancer. AMD3100 also mobilizes most mature leukocyte subsets to blood; however, their source and trafficking potential have not been fully delineated. Here, we show that a single injection of AMD3100 10 mg/kg into C57Bl/6 mice rapidly mobilizes (peak ∼ 2.5 h) the same leukocyte subsets to blood as in humans. Using this model, we found that AMD3100 mobilization of neutrophils, lymphocytes, and monocytes to blood is not reduced by splenectomy or by blockade of lymphocyte egress from lymph node with FTY720, but is coupled to (i) reduced content of each of these cell types in the bone marrow; (ii) reduced T-cell numbers in thymuses; (iii) increased lymphocytes in lymph nodes; and (iv) increased neutrophil and monocyte content in the lung. Direct intrathymic labeling showed that AMD3100 selectively mobilizes naïve thymic CD4(+) and CD8(+) T cells to blood. Finally, AMD3100-induced neutrophil mobilization to blood did not reduce neutrophil trafficking to thioglycollate-inflamed peritoneum. Thus, AMD3100 redistributes lymphocytes, monocytes, and neutrophils from primary immune organs to secondary immune organs, peripheral tissues, and blood, without compromising neutrophil trafficking to inflamed sites.

Human monocyte subsets at homeostasis and their perturbation in numbers and function in filarial infection.

To characterize the function and plasticity of the major human circulating monocyte populations and to explore their role in systemic helminth infection, highly purified (by flow-based sorting) human monocyte subsets (CD14(hi)/CD16(neg) [classical], CD14(+ or hi)/CD16(med) [intermediate], and CD14(neg)/CD16(hi) [nonclassical]) were examined at homeostasis and after activation. Among these three subsets the classical and intermediate subsets were found to be the major sources of inflammatory and regulatory cytokines, as well as cytokines/chemokines associated with alternative activation, whereas the nonclassical and classical populations demonstrated an ability to transmigrate through endothelial monolayers. Moreover, it was primarily the classical subset that was the most efficient in promoting autologous T cell proliferation. The distribution of these subsets changed in the context of a systemic helminth (Wuchereria bancrofti) infection such that patent infection altered the frequency and distribution of these monocyte subsets with the nonclassical monocytes being expanded (almost 2-fold) in filarial infection. To understand further the filarial/monocyte interface, in vitro modeling demonstrated that the classical subset internalized filarial antigens more efficiently than the other two subsets but that the parasite-driven regulatory cytokine interleukin-10 was exclusively coming from the intermediate subset. Our data suggest that monocyte subsets have a differential function at homeostasis and in response to helminth parasites.