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1.
mBio ; 15(1): e0022523, 2024 Jan 16.
Article in English | MEDLINE | ID: mdl-38112465

ABSTRACT

IMPORTANCE: The prevalence of multidrug-resistant Staphylococcus aureus is of global concern, and vaccines are urgently needed. The iron-regulated surface determinant protein B (IsdB) of S. aureus was investigated as a vaccine candidate because of its essential role in bacterial iron acquisition but failed in clinical trials despite strong immunogenicity. Here, we reveal an unexpected second function for IsdB in pathogen-host interaction: the bacterial fitness factor IsdB triggers a strong inflammatory response in innate immune cells via Toll-like receptor 4 and the inflammasome, thus acting as a novel pathogen-associated molecular pattern of S. aureus. Our discovery contributes to a better understanding of how S. aureus modulates the immune response, which is necessary for vaccine development against the sophisticated pathogen.


Subject(s)
Bacterial Proteins , Cation Transport Proteins , Cytokines , Methicillin-Resistant Staphylococcus aureus , NLR Family, Pyrin Domain-Containing 3 Protein , Staphylococcal Infections , Toll-Like Receptor 4 , Humans , Bacterial Proteins/immunology , Caspase 1/metabolism , Cation Transport Proteins/immunology , Cytokines/metabolism , Inflammasomes/metabolism , Iron/metabolism , Methicillin-Resistant Staphylococcus aureus/immunology , NLR Family, Pyrin Domain-Containing 3 Protein/genetics , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Staphylococcal Infections/immunology , Toll-Like Receptor 4/metabolism
2.
J Mol Biol ; 435(17): 168192, 2023 09 01.
Article in English | MEDLINE | ID: mdl-37394032

ABSTRACT

CorA, the primary magnesium ion channel in prokaryotes and archaea, is a prototypical homopentameric ion channel that undergoes ion-dependent conformational transitions. CorA adopts five-fold symmetric non-conductive states in the presence of high concentrations of Mg2+, and highly asymmetric flexible states in its complete absence. However, the latter were of insufficient resolution to be thoroughly characterized. In order to gain additional insights into the relationship between asymmetry and channel activation, we exploited phage display selection strategies to generate conformation-specific synthetic antibodies (sABs) against CorA in the absence of Mg2+. Two sABs from these selections, C12 and C18, showed different degrees of Mg2+-sensitivity. Through structural, biochemical, and biophysical characterization, we found the sABs are both conformation-specific but probe different features of the channel under open-like conditions. C18 is highly specific to the Mg2+-depleted state of CorA and through negative-stain electron microscopy (ns-EM), we show sAB binding reflects the asymmetric arrangement of CorA protomers in Mg2+-depleted conditions. We used X-ray crystallography to determine a structure at 2.0 Å resolution of sAB C12 bound to the soluble N-terminal regulatory domain of CorA. The structure shows C12 is a competitive inhibitor of regulatory magnesium binding through its interaction with the divalent cation sensing site. We subsequently exploited this relationship to capture and visualize asymmetric CorA states in different [Mg2+] using ns-EM. We additionally utilized these sABs to provide insights into the energy landscape that governs the ion-dependent conformational transitions of CorA.


Subject(s)
Antibodies , Bacterial Proteins , Cation Transport Proteins , Ion Channels , Magnesium , Bacterial Proteins/chemistry , Bacterial Proteins/immunology , Ion Channels/chemistry , Ion Channels/immunology , Magnesium/chemistry , Magnesium/metabolism , Protein Conformation , Cation Transport Proteins/chemistry , Cation Transport Proteins/immunology , Antibodies/chemistry
3.
Int J Mol Sci ; 22(15)2021 Jul 27.
Article in English | MEDLINE | ID: mdl-34360779

ABSTRACT

Pro-inflammatory cytokines promote cellular iron-import through enhanced divalent metal transporter-1 (DMT1) expression in pancreatic ß-cells, consequently cell death. Inhibition of ß-cell iron-import by DMT1 silencing protects against apoptosis in animal models of diabetes. However, how alterations of signaling networks contribute to the protective action of DMT1 knock-down is unknown. Here, we performed phosphoproteomics using our sequential enrichment strategy of mRNA, protein, and phosphopeptides, which enabled us to explore the concurrent molecular events in the same set of wildtype and DMT1-silenced ß-cells during IL-1ß exposure. Our findings reveal new phosphosites in the IL-1ß-induced proteins that are clearly reverted by DMT1 silencing towards their steady-state levels. We validated the levels of five novel phosphosites of the potential protective proteins using parallel reaction monitoring. We also confirmed the inactivation of autophagic flux that may be relevant for cell survival induced by DMT1 silencing during IL-1ß exposure. Additionally, the potential protective proteins induced by DMT1 silencing were related to insulin secretion that may lead to improving ß-cell functions upon exposure to IL-1ß. This global profiling has shed light on the signal transduction pathways driving the protection against inflammation-induced cell death in ß-cells after DMT1 silencing.


Subject(s)
Apoptosis/immunology , Autophagy/immunology , Cation Transport Proteins/deficiency , Gene Knockdown Techniques , Insulin-Secreting Cells/immunology , Interleukin-1beta/immunology , Interleukin-6/immunology , Signal Transduction/immunology , Animals , Apoptosis/genetics , Autophagy/genetics , Cation Transport Proteins/immunology , Interleukin-1beta/genetics , Interleukin-6/genetics , Mice , Signal Transduction/genetics
4.
Front Immunol ; 12: 684823, 2021.
Article in English | MEDLINE | ID: mdl-34122448

ABSTRACT

HI, a fusion protein that consists of the alpha-toxin (Hla) and the N2 domain of iron surface determinant B (IsdB), is one of the antigens in the previously reported S. aureus vaccine rFSAV and has already entered phase II clinical trials. Previous studies revealed that HI is highly immunogenic in both mice and healthy volunteers, and the humoral immune response plays key roles in HI-mediated protection. In this study, we further investigated the protective efficacy of immunization with HI plus four different adjuvants in a mouse bacteremia model. Results showed that HI-mediated protection was altered in response to different adjuvants. Using antisera from immunized mice, we identified seven B-cell immunodominant epitopes on Hla and IsdB, including 6 novel epitopes (Hla1-18, Hla84-101, Hla186-203, IsdB342-359, IsdB366-383, and IsdB384-401). The immunodominance of B-cell epitopes, total IgG titers and the levels of IFN-γ and IL-17A from mice immunized with HI plus different adjuvants were different from each other, which may explain the difference in protective immunity observed in each immunized group. Thus, our results indicate that adjuvants largely affected the immunodominance of epitopes and the protective efficacy of HI, which may guide further adjuvant screening for vaccine development and optimization.


Subject(s)
Bacteremia/immunology , Bacterial Toxins/immunology , Cation Transport Proteins/immunology , Epitopes, B-Lymphocyte/immunology , Hemolysin Proteins/immunology , Immunodominant Epitopes/immunology , Staphylococcal Infections/prevention & control , Animals , Bacteremia/prevention & control , Disease Models, Animal , Female , Immunization, Passive , Immunotherapy, Adoptive , Interferon-gamma/metabolism , Interleukin-17/metabolism , Mice , Mice, Inbred BALB C , Staphylococcal Infections/immunology , Staphylococcal Vaccines/administration & dosage , Staphylococcal Vaccines/immunology
5.
Vet Immunol Immunopathol ; 235: 110235, 2021 May.
Article in English | MEDLINE | ID: mdl-33838543

ABSTRACT

The aim of this study was to identify virulence factors that have high immunogenicity. An in vivo-expressed Staphylococcus aureus antigen was identified by probing bacteriophage expression libraries of S. aureus with antibodies in bovine mastitis milk. Eighteen clones were isolated, and their proteins were identified as 5 characterised proteins (IsdA, Protein A, IsdB, autolysin, and imidazole glycerol phosphate dehydratase) and 13 hypothetical proteins. We focused on IsdA, IsdB, and IsdH as virulence factors that have a high immunogenicity and are capable of inducing a specific humoral immune response in S. aureus-infected quarters. The optical density (OD) values of IsdA and IsdB IgA and IgG antibodies in milk affected by naturally occurring mastitis caused by S. aureus increased significantly compared to those in healthy milk. In the experimental infection study, the OD values of IsdA- and B-specific IgA and IgG antibodies were significantly increased from 2 to 4 weeks after S. aureus infection compared to day 0 (P < 0.05). On the other hand, we demonstrated that milk from natural and experimental intramammary infections caused by S. aureus are associated with significantly higher IgA levels against IsdH (P < 0.05), but no significant change in IgG levels. Our findings facilitated our understanding of the pathogenicity of S. aureus in bovine mastitis, as well as the mechanisms by which specific humoral immune responses to S. aureus infection are induced. In addition, the results obtained could provide insight into how bovine mastitis can be controlled, for example, through vaccination.


Subject(s)
Antibodies, Bacterial/analysis , Antigens, Bacterial/immunology , Immunoglobulin A/immunology , Mastitis, Bovine/immunology , Mastitis, Bovine/microbiology , Milk/immunology , Staphylococcus aureus/immunology , Animals , Antibodies, Bacterial/immunology , Antigens, Bacterial/classification , Cation Transport Proteins/immunology , Cattle , Cattle Diseases/immunology , Cattle Diseases/microbiology , Female , Immunity, Humoral , Immunoglobulin A/analysis , Receptors, Cell Surface/immunology
6.
mBio ; 12(1)2021 02 09.
Article in English | MEDLINE | ID: mdl-33563837

ABSTRACT

Nrf2 is a redox-sensitive transcription factor that is thought to be important in protection against intracellular pathogens. To determine the protective role of Nrf2 in the host defense against Mycobacterium avium complex (MAC), both wild-type and Nrf2-deficient mice were intranasally infected with MAC bacteria. Nrf2-deficient mice were highly susceptible to MAC bacteria compared with wild-type mice. There were no significant changes in the levels of oxidative stress and Th1 cytokine production between genotypes. Comprehensive transcriptome analysis showed that the expressions of Nramp1 and HO-1 were much lower in the infected lungs, and the expression of Nramp1 was especially lower in alveolar macrophages of Nrf2-deficient mice than of wild-type mice. Electron microscopy showed that many infected alveolar macrophages from Nrf2-deficient mice contained a large number of intracellular MAC bacteria with little formation of phagolysosomes, compared with those from wild-type mice. Treatment with sulforaphane, an activator of Nrf2, increased resistance to MAC with increased lung expression of Nramp1 and HO-1 in wild-type mice. These results indicate that Nramp1 and HO-1, regulated by Nrf2, are essential in defending against MAC infection due to the promotion of phagolysosome fusion and granuloma formation, respectively. Thus, Nrf2 is thought to be a critical determinant of host resistance to MAC infection.IMPORTANCE Nontuberculous mycobacteria (NTM) are an important cause of morbidity and mortality in pulmonary infections. Among them, Mycobacterium avium complex (MAC) is the most common cause of pulmonary NTM disease worldwide. It is thought that both environmental exposure and host susceptibility are required for the establishment of pulmonary MAC disease, because pulmonary MAC diseases are most commonly observed in slender, postmenopausal women without a clearly recognized immunodeficiency. However, host factors that regulate MAC susceptibility have not been elucidated until now. This study shows that Nrf2 is a critical regulator of host susceptibility to pulmonary MAC disease by promoting phagolysosome fusion and granuloma formation via activating Nramp1 and HO-1 genes, respectively. The Nrf2 system is activated in alveolar macrophages, the most important cells during MAC infection, as both the main reservoir of infection and bacillus-killing cells. Thus, augmentation of Nrf2 might be a useful therapeutic approach for protection against pulmonary MAC disease.


Subject(s)
Cation Transport Proteins/genetics , Gene Expression Regulation/immunology , Granuloma/microbiology , Heme Oxygenase-1/genetics , Host Microbial Interactions , Membrane Proteins/genetics , NF-E2-Related Factor 2/genetics , Animals , Cation Transport Proteins/immunology , Female , Granuloma/immunology , Heme Oxygenase-1/immunology , Host Microbial Interactions/genetics , Host Microbial Interactions/immunology , Macrophage Activation/immunology , Macrophages, Alveolar/immunology , Macrophages, Alveolar/microbiology , Membrane Proteins/immunology , Mice , Mice, Inbred BALB C , Mycobacterium avium Complex/immunology , NF-E2-Related Factor 2/immunology , Oxidative Stress
7.
J Leukoc Biol ; 109(2): 287-297, 2021 02.
Article in English | MEDLINE | ID: mdl-32441444

ABSTRACT

TLR-inducible zinc toxicity is an antimicrobial mechanism utilized by macrophages, however knowledge of molecular mechanisms mediating this response is limited. Here, we show that E. coli exposed to zinc stress within primary human macrophages reside in membrane-bound vesicular compartments. Since SLC30A zinc exporters can deliver zinc into the lumen of vesicles, we examined LPS-regulated mRNA expression of Slc30a/SLC30A family members in primary mouse and human macrophages. A number of these transporters were dynamically regulated in both cell populations. In human monocyte-derived macrophages, LPS strongly up-regulated SLC30A1 mRNA and protein expression. In contrast, SLC30A1 was not LPS-inducible in macrophage-like PMA-differentiated THP-1 cells. We therefore ectopically expressed SLC30A1 in these cells, finding that this was sufficient to promote zinc-containing vesicle formation. The response was similar to that observed following LPS stimulation. Ectopically expressed SLC30A1 localized to both the plasma membrane and intracellular zinc-containing vesicles within LPS-stimulated THP-1 cells. Inducible overexpression of SLC30A1 in THP-1 cells infected with the Escherichia coli K-12 strain MG1655 augmented the zinc stress response of intracellular bacteria and promoted clearance. Furthermore, in THP-1 cells infected with an MG1655 zinc stress reporter strain, all bacteria contained within SLC30A1-positive compartments were subjected to zinc stress. Thus, SLC30A1 marks zinc-containing compartments associated with TLR-inducible zinc toxicity in human macrophages, and its ectopic over-expression is sufficient to initiate this antimicrobial pathway in these cells. Finally, SLC30A1 silencing did not compromise E. coli clearance by primary human macrophages, suggesting that other zinc exporters may also contribute to the zinc toxicity response.


Subject(s)
Cation Transport Proteins/immunology , Escherichia coli Infections/immunology , Macrophages/immunology , Zinc/immunology , Animals , Escherichia coli/immunology , Humans , Lipopolysaccharides/immunology , Macrophages/microbiology , Mice
8.
Trends Microbiol ; 29(2): 98-106, 2021 02.
Article in English | MEDLINE | ID: mdl-32807623

ABSTRACT

Host organisms utilize nutritional immunity to limit the availability of nutrients essential to an invading pathogen. Nutrients may include amino acids, nucleotide bases, and transition metals, the essentiality of which varies among pathogens. The mammalian macrophage protein Slc11a1 (previously Nramp1) mediates resistance to several intracellular pathogens. Slc11a1 is proposed to restrict growth of Salmonella enterica serovar Typhimurium in host tissues by causing magnesium deprivation. This is intriguing because magnesium is the most abundant divalent cation in all living cells. A pathogen's response to factors such as Slc11a1 that promote nutritional immunity may therefore reflect what the pathogen 'feels' in its cytoplasm, rather than the nutrient concentration in host cell compartments.


Subject(s)
Macrophages/immunology , Magnesium/metabolism , Salmonella Infections/immunology , Animals , Cation Transport Proteins/genetics , Cation Transport Proteins/immunology , Host-Pathogen Interactions , Humans , Macrophages/microbiology , Magnesium/immunology , Salmonella Infections/metabolism , Salmonella Infections/microbiology , Salmonella Infections/physiopathology , Salmonella typhimurium/genetics , Salmonella typhimurium/metabolism
9.
JCI Insight ; 5(19)2020 10 02.
Article in English | MEDLINE | ID: mdl-33004694

ABSTRACT

Staphylococcus aureus is prevalent in surgical site infections (SSI) and leads to death in approximately 1% of patients. Phase IIB/III clinical trial results have demonstrated that vaccination against the iron-regulated surface determinant protein B (IsdB) is associated with an increased mortality rate in patients with SSI. Thus, we hypothesized that S. aureus induces nonneutralizing anti-IsdB antibodies, which facilitate bacterial entry into leukocytes to generate "Trojan horse" leukocytes that disseminate the pathogen. Since hemoglobin (Hb) is the primary target of IsdB, and abundant Hb-haptoglobin (Hb-Hp) complexes in bleeding surgical wounds are normally cleared via CD163-mediated endocytosis by macrophages, we investigated this mechanism in vitro and in vivo. Our results demonstrate that active and passive IsdB immunization of mice renders them susceptible to sepsis following SSI. We also found that a multimolecular complex containing S. aureus protein A-anti-IsdB-IsdB-Hb-Hp mediates CD163-dependent bacterial internalization of macrophages in vitro. Moreover, IsdB-immunized CD163-/- mice are resistant to sepsis following S. aureus SSI, as are normal healthy mice given anti-CD163-neutralizing antibodies. These genetic and biologic CD163 deficiencies did not exacerbate local infection. Thus, anti-IsdB antibodies are a risk factor for S. aureus sepsis following SSI, and disruption of the multimolecular complex and/or CD163 blockade may intervene.


Subject(s)
Antibodies, Bacterial/adverse effects , Antibodies, Monoclonal/adverse effects , Cation Transport Proteins/immunology , Sepsis/etiology , Staphylococcal Infections/complications , Staphylococcus aureus/immunology , Surgical Wound Infection/complications , Animals , Antigens, CD/immunology , Antigens, CD/metabolism , Antigens, Differentiation, Myelomonocytic/immunology , Antigens, Differentiation, Myelomonocytic/metabolism , Female , Haptoglobins/immunology , Haptoglobins/metabolism , Hemoglobins/immunology , Hemoglobins/metabolism , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Receptors, Cell Surface/immunology , Receptors, Cell Surface/metabolism , Sepsis/metabolism , Sepsis/pathology , Staphylococcal Infections/immunology , Staphylococcal Infections/microbiology , Surgical Wound Infection/immunology , Surgical Wound Infection/microbiology
10.
PLoS One ; 15(7): e0235776, 2020.
Article in English | MEDLINE | ID: mdl-32645059

ABSTRACT

Macrophages are key phagocytic cells and play an important role in eliminating external microorganisms and endogenous danger signals. Dysregulation in macrophage functions have been reported in patients with asthma. Zinc homeostasis is critical in maintaining macrophage functions. The solute carrier (SLC) protein SLC39A7, a Zn2+ importer, has recently been linked to asthma. However, the roles of SLC39A7 in macrophage phagocytosis are not well understood. Here we found that phagocytosis efficiency was significantly decreased in SLC39A7-knockdown THP-1 cells, however the phagocytosis capability could be reversed with zinc supplementation. SLC39A7 deficiency skewed macrophages towards alternative activation, as indicated by increased expression of M2 activation marker CD206 and decreased expression of M1 activation marker NOS2. Consistent to this result, SLC39A7-knockdown cells produced reduced amounts of proinflammatory cytokines TNF- and IL-6. Furthermore, the mRNA level of receptor Clec4e previously known to be involved in phagocytosis of BCG was significantly reduced in SLC39A7 knockdown cells. Importantly, all these defects due to SLC39A7 deficiency could be reversed by zinc supplementation. Thus, zinc transporter SLC39A7 provide support for phagocytosis and classical macrophage activation.


Subject(s)
Cation Transport Proteins/immunology , Macrophage Activation , Phagocytosis , Zinc/deficiency , Cell Line , Humans , Macrophages/immunology , Zinc/immunology
11.
PLoS One ; 15(7): e0235563, 2020.
Article in English | MEDLINE | ID: mdl-32645092

ABSTRACT

Western blotting has been widely used for investigation of protein expression, posttranslational modifications, and interactions. Because western blotting usually involves heat-denaturation of samples prior to gel loading, clarification of detailed procedures for sample preparation have been omitted or neglected in many publications. We show here the case that even excellent primary antibodies failed to detect a specific protein of interest due to a routine heating practice of protein samples. We performed western blotting for transmembrane iron transporter proteins; SLC11A2 (divalent metal transporter 1, DMT1), SLC40A1 (ferroportin 1, Fpn1), and transferrin receptor-1 (TfR1), along with cytoplasmic iron storage protein ferritin H. Our results in 12 human culture cell lysates indicated that only unheated samples prior to gel loading gave rise to clear resolution of DMT1 protein, while heated samples (95°C, 5min) caused the loss of resolution due to DMT1 protein aggregates. Unheated samples also resulted in better resolution for Fpn1 and TfR1 western blots. Conversely, only heated samples allowed to detect ferritin H, otherwise ferritin polymers failed to get into the gel. Neither different lysis/sample loading buffers nor sonication improved the resolution of DMT1 and Fpn1 western blots. Thus, heating samples most critically affected the outcome of western blotting, suggesting the similar cases for thousands of other transmembrane and heat-sensitive proteins.


Subject(s)
Blotting, Western/methods , Cation Transport Proteins/analysis , Cell Fractionation/methods , A549 Cells , Antibodies/immunology , Caco-2 Cells , Cation Transport Proteins/immunology , HEK293 Cells , HeLa Cells , Hep G2 Cells , Humans , Jurkat Cells , Limit of Detection , MCF-7 Cells
12.
Cell Host Microbe ; 27(3): 454-466.e8, 2020 Mar 11.
Article in English | MEDLINE | ID: mdl-32075740

ABSTRACT

Type I interferons (IFNs-I) fulfil multiple protective functions during pathogenic infections, but they can also cause detrimental effects and enhance immunopathology. Here, we report that IFNs-I promote the dysregulation of iron homeostasis in macrophages during systemic infections with the intracellular pathogen Candida glabrata, leading to fungal survival and persistence. By engaging JAK1, IFNs-I disturb the balance of the transcriptional activator NRF2 and repressor BACH1 to induce downregulation of the key iron exporter Fpn1 in macrophages. This leads to enhanced iron accumulation in the phagolysosome and failure to restrict fungal access to iron pools. As a result, C. glabrata acquires iron via the Sit1/Ftr1 iron transporter system, facilitating fungal intracellular replication and immune evasion. Thus, IFNs-I are central regulators of iron homeostasis, which can impact infection, and restricting iron bioavailability may offer therapeutic strategies to combat invasive fungal infections.


Subject(s)
Candida glabrata/pathogenicity , Homeostasis , Interferon Type I/immunology , Iron/physiology , Macrophages/microbiology , Adult , Animals , Basic-Leucine Zipper Transcription Factors/immunology , Candidiasis/immunology , Cation Transport Proteins/immunology , Cells, Cultured , Female , Humans , Immune Evasion , Janus Kinase 1/immunology , Macrophages/immunology , Male , Mice, Inbred C57BL , Mice, Knockout , NF-E2-Related Factor 2/immunology , Phagosomes/microbiology , Spleen/immunology
13.
J Clin Invest ; 130(1): 507-522, 2020 01 02.
Article in English | MEDLINE | ID: mdl-31714901

ABSTRACT

X-linked immunodeficiency with magnesium defect, EBV infection, and neoplasia (XMEN) disease are caused by deficiency of the magnesium transporter 1 (MAGT1) gene. We studied 23 patients with XMEN, 8 of whom were EBV naive. We observed lymphadenopathy (LAD), cytopenias, liver disease, cavum septum pellucidum (CSP), and increased CD4-CD8-B220-TCRαß+ T cells (αßDNTs), in addition to the previously described features of an inverted CD4/CD8 ratio, CD4+ T lymphocytopenia, increased B cells, dysgammaglobulinemia, and decreased expression of the natural killer group 2, member D (NKG2D) receptor. EBV-associated B cell malignancies occurred frequently in EBV-infected patients. We studied patients with XMEN and patients with autoimmune lymphoproliferative syndrome (ALPS) by deep immunophenotyping (32 immune markers) using time-of-flight mass cytometry (CyTOF). Our analysis revealed that the abundance of 2 populations of naive B cells (CD20+CD27-CD22+IgM+HLA-DR+CXCR5+CXCR4++CD10+CD38+ and CD20+CD27-CD22+IgM+HLA-DR+CXCR5+CXCR4+CD10-CD38-) could differentially classify XMEN, ALPS, and healthy individuals. We also performed glycoproteomics analysis on T lymphocytes and show that XMEN disease is a congenital disorder of glycosylation that affects a restricted subset of glycoproteins. Transfection of MAGT1 mRNA enabled us to rescue proteins with defective glycosylation. Together, these data provide new clinical and pathophysiological foundations with important ramifications for the diagnosis and treatment of XMEN disease.


Subject(s)
Autoimmune Lymphoproliferative Syndrome/immunology , Magnesium Deficiency/immunology , X-Linked Combined Immunodeficiency Diseases/immunology , Antigens, CD/genetics , Antigens, CD/immunology , Autoimmune Lymphoproliferative Syndrome/genetics , Autoimmune Lymphoproliferative Syndrome/pathology , CD4-CD8 Ratio , Cation Transport Proteins/genetics , Cation Transport Proteins/immunology , Female , Glycosylation , Humans , Magnesium Deficiency/genetics , Magnesium Deficiency/pathology , Male , X-Linked Combined Immunodeficiency Diseases/genetics , X-Linked Combined Immunodeficiency Diseases/pathology
14.
J Leukoc Biol ; 106(5): 1079-1088, 2019 11.
Article in English | MEDLINE | ID: mdl-31166618

ABSTRACT

The intestinal microbiota has several effects on host physiology. Previous work from our laboratory demonstrated that the microbiota influences systemic iron homeostasis in mouse colitis models by altering inflammation-induced expression of the iron-regulating hormone hepcidin. In the present study, we examined the impact of the gut commensal bacterium Bacteroides fragilis on the expression of the iron exporter ferroportin, the target of hepcidin action, in macrophages, the cell type that plays a pivotal role in iron recycling. Mouse bone marrow-derived macrophages were exposed to B. fragilis and were analyzed by quantitative real-time polymerase chain reaction and Western blotting. We found that B. fragilis down-regulated ferroportin transcription independently of bacterial viability. Medium conditioned by the bacteria also reduced ferroportin expression, indicating the involvement of soluble factors, possibly Toll-like receptor ligands. Consistent with this idea, several of these ligands were able to down-regulate ferroportin. The B. fragilis-induced decrease in ferroportin was functionally important since it produced a significant increase in intracellular iron concentrations that prevented the effects of the iron chelator deferoxamine on Salmonella-induced IL-6 and IL-1ß production. Our results thus reveal that B. fragilis can influence macrophage iron handling and inflammatory responses by modulating ferroportin expression.


Subject(s)
Bacteroides fragilis/immunology , Cation Transport Proteins/immunology , Down-Regulation/immunology , Homeostasis/immunology , Iron/immunology , Macrophages/immunology , Animals , Macrophages/microbiology , Mice
15.
Parasitol Res ; 118(8): 2329-2342, 2019 Aug.
Article in English | MEDLINE | ID: mdl-31230160

ABSTRACT

Leishmaniases are cutaneous, mucocutaneous, and visceral diseases affecting humans and domesticated animals mostly in the tropical and subtropical areas of the planet. Host genetics have been widely investigated for their role in developing various infectious diseases. The SLC11A1 gene has been reported to play a role in neutrophil function and is associated with susceptibility to infectious and inflammatory diseases such as tuberculosis or rheumatoid arthritis. In the present meta-analysis, we investigate the genetic association of SLC11A1 polymorphisms with susceptibility to leishmaniasis. Genotypes and other risk-related data were collected from 13 case-control and family-based studies (after literature search). Conventional random-effects meta-analysis was performed using STATA 13. To pool case-control and family-based data, the weighted Stouffer's method was also applied. Eight polymorphisms were investigated: rs2276631, rs3731865, rs3731864, rs17221959, rs201565523, rs2279015, rs17235409, and rs17235416. We found that rs17235409 (D543N) and rs17235416 (1729 + 55del4) are significantly associated with a risk for cutaneous leishmaniasis (CL), whereas rs17221959, rs2279015, and rs17235409 are associated with visceral leishmaniasis (VL). Our results suggest that polymorphisms in SLC11A1 affect susceptibility to CL and VL. These findings open new pathways in understanding macrophage response to Leishmania infection and the genetic factors predisposing to symptomatic CL or VL that can lead to the usage of predictive biomarkers in populations at risk.


Subject(s)
Cation Transport Proteins/genetics , Leishmaniasis, Cutaneous/genetics , Leishmaniasis, Visceral/genetics , Polymorphism, Single Nucleotide , Case-Control Studies , Cation Transport Proteins/immunology , Genetic Predisposition to Disease , Genotype , Humans , Leishmania/physiology , Leishmaniasis, Cutaneous/immunology , Leishmaniasis, Cutaneous/parasitology , Leishmaniasis, Visceral/immunology , Leishmaniasis, Visceral/parasitology , Macrophages/immunology , Neutrophils/immunology , STAT Transcription Factors/genetics , STAT Transcription Factors/immunology
16.
Indian J Med Res ; 149(1): 34-40, 2019 Jan.
Article in English | MEDLINE | ID: mdl-31115372

ABSTRACT

BACKGROUND & OBJECTIVES: : Sickle cell disease (SCD) patients require red cell transfusion during different clinical complications of the disease. Such patients are at a high risk for developing alloantibody against red cell antigens. From India, there are limited data available on alloantibody formation in multiply transfused SCD patients. The present study was thus undertaken to fill up this lacunae by looking at the development of red cell alloantibodies in SCD and ß-thalassaemia patients on regular transfusion. METHODS: : All sickle cell disease patients undergoing red cell transfusion between 2008 and 2016, were included. During this period, a large number of ß-thalassaemia major patients also underwent regular red cell transfusion. These thalassaemia patients were also included to compare the tendency of antibody formation between SCD and ß-thalassaemia major patients. All patients before regular transfusion were regularly assessed for the development of red cell antibody. Red cell antigen, antibody screen crossmatch and antibody identification were done using the standard technique. RESULTS: : A total of 138 patients with SCD aged between 4 and 53 yr (mean 17.6 yr) consisting of 83 males and 55 females (male:female, 1.5:1) along with 333 transfusion-dependent ß-thalassaemia patients were studied. Over the last eight years, 15 patients with SCD and four patients with thalassaemia developed alloantibody (P <0.001). Antibody specificity of their alloantibodies was against Rhc, RhE, Kell, Fya and Fyb only. Sickle cell disease patients with and without alloantibody required on the average 11.8 and 8.6 units of red cell concentrate, respectively (P <0.05). INTERPRETATION & CONCLUSIONS: : About 11 per cent of the transfused sickle cells patients developed alloantibodies. The antibody specificity was restricted to Rh, Kell and Duffy blood group systems. Extended antigen matching involving Rh, Kell and Duffy antigens may prevent alloantibody in such patients.


Subject(s)
Anemia, Sickle Cell/blood , Erythrocytes/immunology , Isoantibodies/blood , Thalassemia/blood , Adolescent , Adult , Anemia, Sickle Cell/complications , Anemia, Sickle Cell/immunology , Blood Grouping and Crossmatching , Cation Transport Proteins/blood , Cation Transport Proteins/immunology , Child , Child, Preschool , Duffy Blood-Group System/blood , Duffy Blood-Group System/immunology , Erythrocyte Transfusion/methods , Female , Humans , Immunization , Isoantibodies/immunology , Kell Blood-Group System/blood , Kell Blood-Group System/immunology , Male , Membrane Glycoproteins/blood , Membrane Glycoproteins/immunology , Middle Aged , Platelet Transfusion , Receptors, Cell Surface/blood , Receptors, Cell Surface/immunology , Thalassemia/complications , Thalassemia/immunology , Young Adult
17.
Monoclon Antib Immunodiagn Immunother ; 38(2): 70-74, 2019 Apr.
Article in English | MEDLINE | ID: mdl-31009334

ABSTRACT

Zinc transporter ZIP6 (SLC39A6) or LIV-1 is a protein that belongs to a subfamily of proteins group that displays structural specifications of zinc transporters in the cell membrane. Overexpression of this protein is observed in breast, prostate, and kidney tumor cells. Lately, LIV-1 is a dependable marker for detection of estrogen receptor positive breast cancer, which can be used to detect luminal breast cancer type A. In this study, the gene construct containing extracellular domain of human LIV-1 gene was subcloned into pET22b expression vector, expressed and confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and western blotting. It was shown for the first time that the extracellular domain of LIV-1 could be expressed in bacterial systems and can be used for rabbit immunization. The reactivity of the resulted antibody was evaluated in flow cytometry and enzyme-linked immunosorbent assay. In conclusion, this protein can be used for animal immunization toward preparation of a new monoclonal antibody that can be introduced as a drug in the treatment of breast cancer.


Subject(s)
Antibodies, Monoclonal/biosynthesis , Antibodies, Monoclonal/immunology , Antibody Formation , Breast Neoplasms/metabolism , Cation Transport Proteins/immunology , Neoplasm Proteins/immunology , Recombinant Proteins/immunology , Animals , Breast Neoplasms/immunology , Female , Humans , Rabbits , Tumor Cells, Cultured
18.
Nat Immunol ; 20(3): 350-361, 2019 03.
Article in English | MEDLINE | ID: mdl-30718914

ABSTRACT

Despite the known importance of zinc for human immunity, molecular insights into its roles have remained limited. Here we report a novel autosomal recessive disease characterized by absent B cells, agammaglobulinemia and early onset infections in five unrelated families. The immunodeficiency results from hypomorphic mutations of SLC39A7, which encodes the endoplasmic reticulum-to-cytoplasm zinc transporter ZIP7. Using CRISPR-Cas9 mutagenesis we have precisely modeled ZIP7 deficiency in mice. Homozygosity for a null allele caused embryonic death, but hypomorphic alleles reproduced the block in B cell development seen in patients. B cells from mutant mice exhibited a diminished concentration of cytoplasmic free zinc, increased phosphatase activity and decreased phosphorylation of signaling molecules downstream of the pre-B cell and B cell receptors. Our findings highlight a specific role for cytosolic Zn2+ in modulating B cell receptor signal strength and positive selection.


Subject(s)
Agammaglobulinemia/immunology , B-Lymphocytes/immunology , Cation Transport Proteins/immunology , Zinc/immunology , Agammaglobulinemia/genetics , Agammaglobulinemia/metabolism , Animals , B-Lymphocytes/metabolism , Cation Transport Proteins/deficiency , Cation Transport Proteins/genetics , Child, Preschool , Cytosol/immunology , Cytosol/metabolism , Disease Models, Animal , Endoplasmic Reticulum/immunology , Endoplasmic Reticulum/metabolism , Female , Gene Expression Profiling , Humans , Infant , Male , Mice, Inbred C57BL , Mice, Transgenic , Mutation , Pedigree , Zinc/metabolism
19.
J Immunol ; 202(2): 441-450, 2019 01 15.
Article in English | MEDLINE | ID: mdl-30552163

ABSTRACT

Zinc deficiency causes immune dysfunction. In T lymphocytes, hypozincemia promotes thymus atrophy, polarization imbalance, and altered cytokine production. Zinc supplementation is commonly used to boost immune function to prevent infectious diseases in at-risk populations. However, the molecular players involved in zinc homeostasis in lymphocytes are poorly understood. In this paper, we wanted to determine the identity of the transporter responsible for zinc entry into lymphocytes. First, in human Jurkat cells, we characterized the effect of zinc on proliferation and activation and found that zinc supplementation enhances activation when T lymphocytes are stimulated using anti-CD3/anti-CD28 Abs. We show that zinc entry depends on specific pathways to correctly tune the NFAT, NF-κB, and AP-1 activation cascades. Second, we used various human and murine models to characterize the zinc transporter family, Zip, during T cell activation and found that Zip6 was strongly upregulated early during activation. Therefore, we generated a Jurkat Zip6 knockout (KO) line to study how the absence of this transporter affects lymphocyte physiology. We found that although Zip6KO cells showed no altered zinc transport or proliferation under basal conditions, under activation, these KO cells showed deficient zinc transport and a drastically impaired activation program. Our work shows that zinc entry into activated lymphocytes depends on Zip6 and that this transporter is essential for the correct function of the cellular activation machinery.


Subject(s)
Cation Transport Proteins/metabolism , Immunologic Deficiency Syndromes/metabolism , Neoplasm Proteins/metabolism , T-Lymphocytes/immunology , Thymus Gland/pathology , Zinc/metabolism , Animals , Atrophy , Biological Transport , Cation Transport Proteins/genetics , Cation Transport Proteins/immunology , Cell Proliferation , Cytokines/metabolism , Gene Knockdown Techniques , Humans , Jurkat Cells , Lymphocyte Activation , Mice , Mice, Inbred C57BL , Models, Animal , NF-kappa B/metabolism , NFATC Transcription Factors/metabolism , Neoplasm Proteins/genetics , Neoplasm Proteins/immunology , Signal Transduction , Transcription Factor AP-1/metabolism , Up-Regulation
20.
Semin Immunol ; 39: 111-118, 2018 10.
Article in English | MEDLINE | ID: mdl-29950273

ABSTRACT

Leprosy is still a considerable health threat in pockets of several low and middle income countries worldwide where intense transmission is witnessed, and often results in irreversible disabilities and deformities due to delayed- or misdiagnosis. Early detection of leprosy represents a substantial hurdle in present-day leprosy health care. The dearth of timely diagnosis has, however, particularly severe consequences in the case of inflammatory episodes, designated leprosy reactions, which represent the major cause of leprosy-associated irreversible neuropathy. There is currently no accurate, routine diagnostic test to reliably detect leprosy reactions, or to predict which patients will develop these immunological exacerbations. Identification of host biomarkers for leprosy reactions, particularly if correlating with early onset prior to development of clinical symptoms, will allow timely interventions that contribute to decreased morbidity. Development of a point-of-care (POC) test based on such correlates would be a definite game changer in leprosy health care. In this review, proteomic-, transcriptomic and metabolomic research strategies aiming at identification of host biomarker-based correlates of leprosy reactions are discussed, next to external factors associated with occurrence of these episodes. The vast diversity in research strategies combined with the variability in patient- and control cohorts argues for harmonisation of biomarker discovery studies with geographically overarching study sites. This will improve identification of specific correlates associated with risk of these damaging inflammatory episodes in leprosy and subsequent application to rapid field tests.


Subject(s)
Antibodies, Bacterial/analysis , Endpoint Determination/methods , Leprosy/diagnosis , Mycobacterium leprae/immunology , Transcriptome/immunology , Antibodies, Bacterial/biosynthesis , Biomarkers/metabolism , CD30 Ligand/genetics , CD30 Ligand/immunology , Cation Transport Proteins/genetics , Cation Transport Proteins/immunology , Delayed Diagnosis , Disease Progression , Humans , Leprosy/immunology , Leprosy/microbiology , Leprosy/pathology , Metabolome/immunology , Mycobacterium leprae/isolation & purification , Mycobacterium leprae/pathogenicity , Point-of-Care Testing , Systems Biology/methods , Toll-Like Receptors/genetics , Toll-Like Receptors/immunology
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