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1.
J Lipid Res ; 65(2): 100496, 2024 02.
Article in English | MEDLINE | ID: mdl-38185217

ABSTRACT

Pulmonary alveolar proteinosis (PAP) is a life-threatening, rare lung syndrome for which there is no cure and no approved therapies. PAP is a disease of lipid accumulation characterized by alveolar macrophage foam cell formation. While much is known about the clinical presentation, there is a paucity of information regarding temporal changes in lipids throughout the course of disease. Our objectives were to define the detailed lipid composition of alveolar macrophages in PAP patients at the time of diagnosis and during treatment. We performed comprehensive mass spectrometry to profile the lipid signature of alveolar macrophages obtained from three independent mouse models of PAP and from PAP and non-PAP patients. Additionally, we quantified changes in macrophage-associated lipids during clinical treatment of PAP patients. We found remarkable variations in lipid composition in PAP patients, which were consistent with data from three independent mouse models. Detailed lipidomic analysis revealed that the overall alveolar macrophage lipid burden inversely correlated with clinical improvement and response to therapy in PAP patients. Specifically, as PAP patients experienced clinical improvement, there was a notable decrease in the total lipid content of alveolar macrophages. This crucial observation suggests that the levels of these macrophage-associated lipids can be utilized to assess the efficacy of treatment. These findings provide valuable insights into the dysregulated lipid metabolism associated with PAP, offering the potential for lipid profiling to serve as a means of monitoring therapeutic interventions in PAP patients.


Subject(s)
Pulmonary Alveolar Proteinosis , Animals , Mice , Humans , Pulmonary Alveolar Proteinosis/drug therapy , Pulmonary Alveolar Proteinosis/diagnosis , Pulmonary Alveolar Proteinosis/metabolism , Macrophages, Alveolar , Lung/metabolism , Macrophages/metabolism , Lipids
2.
Am J Physiol Lung Cell Mol Physiol ; 325(2): L174-L189, 2023 08 01.
Article in English | MEDLINE | ID: mdl-37366533

ABSTRACT

Pneumonia elicits the production of cytotoxic beta amyloid (Aß) that contributes to end-organ dysfunction, yet the mechanism(s) linking infection to activation of the amyloidogenic pathway that produces cytotoxic Aß is unknown. Here, we tested the hypothesis that gamma-secretase activating protein (GSAP), which contributes to the amyloidogenic pathway in the brain, promotes end-organ dysfunction following bacterial pneumonia. First-in-kind Gsap knockout rats were generated. Wild-type and knockout rats possessed similar body weights, organ weights, circulating blood cell counts, arterial blood gases, and cardiac indices at baseline. Intratracheal Pseudomonas aeruginosa infection caused acute lung injury and a hyperdynamic circulatory state. Whereas infection led to arterial hypoxemia in wild-type rats, the alveolar-capillary barrier integrity was preserved in Gsap knockout rats. Infection potentiated myocardial infarction following ischemia-reperfusion injury, and this potentiation was abolished in knockout rats. In the hippocampus, GSAP contributed to both pre- and postsynaptic neurotransmission, increasing the presynaptic action potential recruitment, decreasing neurotransmitter release probability, decreasing the postsynaptic response, and preventing postsynaptic hyperexcitability, resulting in greater early long-term potentiation but reduced late long-term potentiation. Infection abolished early and late long-term potentiation in wild-type rats, whereas the late long-term potentiation was partially preserved in Gsap knockout rats. Furthermore, hippocampi from knockout rats, and both the wild-type and knockout rats following infection, exhibited a GSAP-dependent increase in neurotransmitter release probability and postsynaptic hyperexcitability. These results elucidate an unappreciated role for GSAP in innate immunity and highlight the contribution of GSAP to end-organ dysfunction during infection.NEW & NOTEWORTHY Pneumonia is a common cause of end-organ dysfunction, both during and in the aftermath of infection. In particular, pneumonia is a common cause of lung injury, increased risk of myocardial infarction, and neurocognitive dysfunction, although the mechanisms responsible for such increased risk are unknown. Here, we reveal that gamma-secretase activating protein, which contributes to the amyloidogenic pathway, is important for end-organ dysfunction following infection.


Subject(s)
Alzheimer Disease , Pneumonia, Bacterial , Rats , Animals , Alzheimer Disease/metabolism , Amyloid Precursor Protein Secretases/genetics , Amyloid Precursor Protein Secretases/metabolism , Multiple Organ Failure , Amyloid beta-Peptides/metabolism , Neurotransmitter Agents
3.
Infect Immun ; 90(3): e0047021, 2022 03 17.
Article in English | MEDLINE | ID: mdl-35130452

ABSTRACT

Pseudomonas aeruginosa is a Gram-negative, opportunistic pathogen that causes nosocomial pneumonia, urinary tract infections, and bacteremia. A hallmark of P. aeruginosa pathogenesis is disruption of host cell function by the type III secretion system (T3SS) and its cognate exoenzyme effectors. The T3SS effector ExoU is phospholipase A2 (PLA2) that targets the host cell plasmalemmal membrane to induce cytolysis and is an important virulence factor that mediates immune avoidance. In addition, ExoU has been shown to subvert the host inflammatory response in a noncytolytic manner. In primary bone marrow-derived macrophages (BMDMs), P. aeruginosa infection is sensed by the nucleotide-binding domain containing leucine-rich repeats-like receptor 4 (NLRC4) inflammasome, which triggers caspase-1 activation and inflammation. ExoU transiently inhibits NLRC4 inflammasome-mediated activation of caspase-1 and its downstream target, interleukin 1ß (IL-1ß), to suppress activation of inflammation. In the present study, we sought to identify additional noncytolytic virulence functions for ExoU and discovered an unexpected association between ExoU, host mitochondria, and NLRC4. We show that infection of BMDMs with P. aeruginosa strains expressing ExoU elicited mitochondrial oxidative stress. In addition, mitochondria and mitochondrion-associated membrane fractions enriched from infected cells exhibited evidence of autophagy activation, indicative of damage. The observation that ExoU elicited mitochondrial stress and damage suggested that ExoU may also associate with mitochondria during infection. Indeed, ExoU phospholipase A2 enzymatic activity was present in enriched mitochondria and mitochondrion-associated membrane fractions isolated from P. aeruginosa-infected BMDMs. Intriguingly, enriched mitochondria and mitochondrion-associated membrane fractions isolated from infected Nlrc4 homozygous knockout BMDMs displayed significantly lower levels of ExoU enzyme activity, suggesting that NLRC4 plays a role in the ExoU-mitochondrion association. These observations prompted us to assay enriched mitochondria and mitochondrion-associated membrane fractions for NLRC4, caspase-1, and IL-1ß. NLRC4 and pro-caspase-1 were detected in enriched mitochondria and mitochondrion-associated membrane fractions isolated from noninfected BMDMs, and active caspase-1 and active IL-1ß were detected in response to P. aeruginosa infection. Interestingly, ExoU inhibited mitochondrion-associated caspase-1 and IL-1ß activation. The implications of ExoU-mediated effects on mitochondria and the NLRC4 inflammasome during P. aeruginosa infection are discussed.


Subject(s)
Pseudomonas Infections , Pseudomonas aeruginosa , Animals , Caspase 1/metabolism , Inflammasomes/metabolism , Inflammation/metabolism , Macrophages/metabolism , Mice , Phospholipases/metabolism , Pseudomonas aeruginosa/physiology , Type III Secretion Systems/metabolism
4.
FASEB J ; 35(9): e21797, 2021 09.
Article in English | MEDLINE | ID: mdl-34383981

ABSTRACT

Pseudomonas aeruginosa is a frequent cause of hospital-acquired lung infections characterized by hyperinflammation, antibiotic resistance, and high morbidity/mortality. Here, we show that the genetic ablation of one cAMP-phosphodiesterase 4 subtype, PDE4B, is sufficient to protect mice from acute lung injury induced by P aeruginosa infection as it reduces pulmonary and systemic levels of pro-inflammatory cytokines, as well as pulmonary vascular leakage and mortality. Surprisingly, despite dampening immune responses, bacterial clearance in the lungs of PDE4B-KO mice is significantly improved compared to WT controls. In wildtypes, P aeruginosa-infection produces high systemic levels of several cytokines, including TNF-α, IL-1ß, and IL-6, that act as cryogens and render the animals hypothermic. This, in turn, diminishes their ability to clear the bacteria. Ablation of PDE4B curbs both the initial production of acute response cytokines, including TNF-α and IL-1ß, as well as their downstream signaling, specifically the induction of the secondary-response cytokine IL-6. This synergistic action protects PDE4B-KO mice from the deleterious effects of the P aeruginosa-induced cytostorm, while concurrently improving bacterial clearance, rather than being immunosuppressive. These benefits of PDE4B ablation are in contrast to the effects resulting from treatment with PAN-PDE4 inhibitors, which have been shown to increase bacterial burden and dissemination. Thus, PDE4B represents a promising therapeutic target in settings of P aeruginosa lung infections.


Subject(s)
Acute Lung Injury/metabolism , Acute Lung Injury/microbiology , Cyclic Nucleotide Phosphodiesterases, Type 4/metabolism , Hypothermia/metabolism , Hypothermia/microbiology , Pseudomonas Infections/metabolism , Pseudomonas aeruginosa/pathogenicity , Animals , Cytokines/metabolism , Lung/metabolism , Lung/microbiology , Mice , Mice, Inbred C57BL , Mice, Knockout , Phosphodiesterase 4 Inhibitors/pharmacology , Pseudomonas Infections/microbiology , Signal Transduction/physiology , Tumor Necrosis Factor-alpha/metabolism
5.
Am J Respir Cell Mol Biol ; 61(2): 198-208, 2019 08.
Article in English | MEDLINE | ID: mdl-30741559

ABSTRACT

We established a murine model of multiwall carbon nanotube (MWCNT)-elicited chronic granulomatous disease that bears similarities to human sarcoidosis pathology, including alveolar macrophage deficiency of peroxisome proliferator-activated receptor γ (PPARγ). Because lymphocyte reactivity to mycobacterial antigens has been reported in sarcoidosis, we hypothesized that addition of mycobacterial ESAT-6 (early secreted antigenic target protein 6) to MWCNT might exacerbate pulmonary granulomatous pathology. MWCNTs with or without ESAT-6 peptide 14 were instilled by the oropharyngeal route into macrophage-specific PPARγ-knockout (KO) or wild-type mice. Control animals received PBS or ESAT-6. Lung tissues, BAL cells, and BAL fluid were evaluated 60 days after instillation. PPARγ-KO mice receiving MWCNT + ESAT-6 had increased granulomas and significantly elevated fibrosis (trichrome staining) compared with wild-type mice or PPARγ-KO mice that received only MWCNT. Immunostaining of lung tissues revealed elevated fibronectin and Siglec F expression on CD11c+ infiltrating alveolar macrophages in the presence of MWCNT + ESAT-6 compared with MWCNT alone. Analyses of BAL fluid proteins indicated increased levels of transforming growth factor (TGF)-ß and the TGF-ß pathway mediator IL-13 in PPARγ-KO mice that received MWCNT + ESAT-6 compared with wild-type or PPARγ-KO mice that received MWCNT. Similarly, mRNA levels of matrix metalloproteinase 9, another requisite factor for TGF-ß production, was elevated in PPARγ-KO mice by MWCNT + ESAT-6. Analysis of ESAT-6 in lung tissues by mass spectrometry revealed ESAT-6 retention in lung tissues of PPARγ-KO but not wild-type mice. These data indicate that PPARγ deficiency promotes pulmonary ESAT-6 retention, exacerbates macrophage responses to MWCNT + ESAT-6, and intensifies pulmonary fibrosis. The present findings suggest that the model may facilitate understanding of the effects of environmental factors on sarcoidosis-associated pulmonary fibrosis.


Subject(s)
Antigens, Bacterial/pharmacology , Bacterial Proteins/pharmacology , Macrophages, Alveolar/metabolism , PPAR gamma/deficiency , Pulmonary Fibrosis/microbiology , Sarcoidosis, Pulmonary/microbiology , Animals , Bronchoalveolar Lavage , Bronchoalveolar Lavage Fluid , CD11 Antigens/metabolism , Disease Models, Animal , Fibronectins/metabolism , Fibrosis/metabolism , Inflammation , Lung/pathology , Macrophages/metabolism , Mass Spectrometry , Mice , Mice, Inbred C57BL , Mice, Knockout , Nanotubes, Carbon/chemistry , PPAR gamma/genetics , Pulmonary Fibrosis/genetics , Sarcoidosis, Pulmonary/pathology
6.
J Immunol ; 197(2): 470-9, 2016 07 15.
Article in English | MEDLINE | ID: mdl-27279372

ABSTRACT

Pulmonary alveolar proteinosis (PAP) is a rare lung syndrome caused by the accumulation of surfactants in the alveoli. The most prevalent clinical form of PAP is autoimmune PAP (aPAP) whereby IgG autoantibodies neutralize GM-CSF. GM-CSF is a pleiotropic cytokine that promotes the differentiation, survival, and activation of alveolar macrophages, the cells responsible for surfactant degradation. IgG-mediated neutralization of GM-CSF thereby inhibits alveolar macrophage homeostasis and function, leading to surfactant accumulation and innate immunodeficiency. Importantly, there are no rodent models for this disease; therefore, underlying immune mechanisms regulating GM-CSF-specific IgG in aPAP are not well understood. In this article, we identify that autoimmune-prone Rasgrp1-deficient mice develop aPAP: 1) Rasgrp1-deficient mice exhibit reduced pulmonary compliance and lung histopathology characteristic of PAP; 2) alveolar macrophages from Rasgrp1-deficient mice are enlarged and exhibit reduced surfactant degradation; 3) the concentration of GM-CSF-specific IgG is elevated in both serum and bronchoalveolar lavage fluid from Rasgrp1-deficient mice; 4) GM-CSF-specific IgG is capable of neutralizing GM-CSF bioactivity; and 5) Rasgrp1-deficient mice also lacking CD275/ICOSL, a molecule necessary for conventional T cell-dependent Ab production, have reduced GM-CSF-specific autoantibody and do not develop PAP. Collectively, these studies reveal that Rasgrp1-deficient mice, to our knowledge, represent the first rodent model for aPAP.


Subject(s)
Autoantibodies/immunology , Autoimmune Diseases/immunology , Disease Models, Animal , Guanine Nucleotide Exchange Factors/deficiency , Pulmonary Alveolar Proteinosis/immunology , Animals , Autoantigens/immunology , Autoimmune Diseases/genetics , Enzyme-Linked Immunosorbent Assay , Granulocyte-Macrophage Colony-Stimulating Factor/immunology , Guanine Nucleotide Exchange Factors/immunology , Immunohistochemistry , Mice , Mice, Inbred C57BL , Mice, Knockout , Pulmonary Alveolar Proteinosis/genetics , Real-Time Polymerase Chain Reaction
7.
J Immunol ; 196(12): 4935-46, 2016 06 15.
Article in English | MEDLINE | ID: mdl-27183569

ABSTRACT

T cells undergo homeostatic expansion and acquire an activated phenotype in lymphopenic microenvironments. Restoration of normal lymphocyte numbers typically re-establishes normal homeostasis, and proinflammatory cytokine production returns to baseline. Mice deficient in guanine nucleotide exchange factor RasGRP1 exhibit dysregulated homeostatic expansion, which manifests as lymphoproliferative disease with autoantibody production. Our previous work revealed that autoreactive B cells lacking RasGRP1 break tolerance early during development, as well as during germinal center responses, suggesting that T cell-independent and T cell-dependent mechanisms are responsible. Examination of whether a particular T cell subset is involved in the breach of B cell tolerance revealed increased Th17 cells in Rasgrp1-deficient mice relative to control mice. Rasgrp1-deficient mice lacking IL-17R had fewer germinal centers, and germinal centers that formed contained fewer autoreactive B cells, suggesting that IL-17 signaling is required for a break in B cell tolerance in germinal centers. Interestingly, a fraction of Th17 cells from Rasgrp1-deficient mice were CXCR5(+) and upregulated levels of CD278 coordinate with their appearance in germinal centers, all attributes of T follicular helper cells (Tfh17). To determine whether CD278-CD275 interactions were required for the development of Tfh17 cells and for autoantibody, Rasgrp1-deficient mice were crossed with CD275-deficient mice. Surprisingly, mice deficient in RasGRP1 and CD275 formed Tfh17 cells and germinal centers and produced similar titers of autoantibodies as mice deficient in only RasGRP1. Therefore, these studies suggest that requirements for Tfh cell development change in lymphopenia-associated autoimmune settings.


Subject(s)
Autoimmunity , Germinal Center/immunology , Inducible T-Cell Co-Stimulator Ligand/immunology , Interleukin-17/immunology , Lymphopenia/immunology , T-Lymphocytes, Helper-Inducer/immunology , Animals , Autoantibodies/biosynthesis , Autoantibodies/immunology , B-Lymphocytes/immunology , Germinal Center/cytology , Guanine Nucleotide Exchange Factors/deficiency , Homeostasis , Immune Tolerance/genetics , Inducible T-Cell Co-Stimulator Ligand/deficiency , Inducible T-Cell Co-Stimulator Protein/genetics , Interleukin-17/biosynthesis , Mice , Receptors, CXCR5/genetics , Receptors, Interleukin-17/deficiency , Signal Transduction , Th17 Cells/immunology
8.
Cell Immunol ; 310: 78-88, 2016 12.
Article in English | MEDLINE | ID: mdl-27502364

ABSTRACT

In activated B cells, increased production of phosphatidylcholine (PtdCho), the most abundant cellular phospholipid, is handled primarily by the CDP-choline pathway. B cell-specific deletion of CTP:phosphocholine cytidylyltransferase α (CCTα), the rate-limiting enzyme in the CDP-choline pathway, led to augmented IgM secretion and reduced IgG production, suggesting that PtdCho synthesis is required for germinal center reactions. To specifically assess whether PtdCho influences B cell fate during germinal center responses, we examined immune responses in mice whereby PtdCho synthesis is disrupted in B cells that have undergone class switch recombination to IgG1 (referred to as either Cγ1wt/wt, Cγ1Cre/wt or Cγ1Cre/Cre based on Cre copy number). Serum IgG1 was markedly reduced in naïve Cγ1Cre/wt and Cγ1Cre/Cre mice, while levels of IgM and other IgG subclasses were similar between Cγ1Cre/wt and Cγ1wt/wt control mice. Serum IgG2b titers were notably reduced and IgG3 titers were increased in Cγ1Cre/Cre mice compared with controls. Following immunization with T cell-dependent antigen NP-KLH, control mice generated high titer IgG anti-NP while IgG anti-NP titers were markedly reduced in both immunized Cγ1Cre/wt and Cγ1Cre/Cre mice. Correspondingly, the frequency of NP-specific IgG antibody-secreting cells was also reduced in spleens and bone marrow of Cγ1Cre/wt and Cγ. 1Cre/Cre mice compared to control mice. Interestingly, though antigen-specific IgM B cells were comparable between Cγ1Cre/wt, Cγ1Cre/Cre and control mice, the frequency and number of IgG1 NP-specific B cells was reduced only in Cγ1Cre/Cre mice. These data indicate that PtdCho is required for the generation of both germinal center-derived B cells and antibody-secreting cells. Further, the reduction in class-switched ASC but not B cells in Cγ1Cre/wt mice suggests that ASC have a greater demand for PtdCho compared to germinal center B cells.


Subject(s)
B-Lymphocytes/immunology , Choline-Phosphate Cytidylyltransferase/metabolism , Phosphatidylcholines/metabolism , Animals , Cell Differentiation/genetics , Cells, Cultured , Choline-Phosphate Cytidylyltransferase/genetics , Germinal Center/pathology , Immunity, Humoral/genetics , Immunoglobulin G/blood , Immunologic Memory/genetics , Lymphocyte Activation , Mice , Mice, Knockout , T-Lymphocytes/immunology , Unfolded Protein Response
9.
J Immunol ; 191(7): 3605-13, 2013 Oct 01.
Article in English | MEDLINE | ID: mdl-23997211

ABSTRACT

Lymphopenic hosts offer propitious microenvironments for expansion of autoreactive B and T cells. Despite this, many lymphopenic hosts do not develop autoimmune disease, suggesting that additional factors are required for breaching self-tolerance in the setting of lymphopenia. Mice deficient in guanine nucleotide exchange factor Rasgrp1 develop a lymphoproliferative disorder with features of human systemic lupus erythematosus. Early in life, Rasgrp1-deficient mice have normal B cell numbers but are T lymphopenic, leading to defective homeostatic expansion of CD4 T cells. To investigate whether B cell-intrinsic mechanisms also contribute to autoimmunity, Rasgrp1-deficient mice were bred to mice containing a knockin autoreactive BCR transgene (564Igi), thereby allowing the fate of autoreactive B cells to be assessed. During B cell development, the frequency of receptor-edited 564Igi B cells was reduced in Rasrp1-deficient mice compared with Rasgrp1-sufficient littermate control mice, suggesting that tolerance was impaired. In addition, the number of 564Igi transitional B cells was increased in Rasgrp1-deficient mice compared with control mice. Immature 564Igi B cells in bone marrow and spleen lacking RasGRP1 expressed lower levels of Bim mRNA and protein, suggesting that autoreactive B cells elude clonal deletion during development. Concomitant with increased serum autoantibodies, Rasgrp1-deficient mice developed spontaneous germinal centers at 8-10 wk of age. The frequency and number of 564Igi B cells within these germinal centers were significantly increased in Rasgrp1-deficient mice relative to control mice. Taken together, these studies suggest that autoreactive B cells lacking Rasgrp1 break central and peripheral tolerance through both T cell-independent and -dependent mechanisms.


Subject(s)
B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Guanine Nucleotide Exchange Factors/genetics , Immune Tolerance/genetics , Animals , Apoptosis Regulatory Proteins/metabolism , Autoantibodies/blood , Autoantibodies/immunology , Autoimmunity/genetics , Autoimmunity/immunology , Bcl-2-Like Protein 11 , Bone Marrow/immunology , Bone Marrow/metabolism , Germinal Center/immunology , Germinal Center/metabolism , Guanine Nucleotide Exchange Factors/deficiency , Membrane Proteins/metabolism , Mice , Mice, Knockout , Proto-Oncogene Proteins/metabolism , Receptors, Antigen, B-Cell/metabolism , Spleen/immunology , Spleen/metabolism , Toll-Like Receptors/metabolism
10.
ERJ Open Res ; 10(1)2024 Jan.
Article in English | MEDLINE | ID: mdl-38410714

ABSTRACT

Background: Sepsis is a life-threatening condition that results from a dysregulated host response to infection, leading to organ dysfunction. Despite the prevalence and associated socioeconomic costs, treatment of sepsis remains limited to antibiotics and supportive care, and a majority of intensive care unit (ICU) survivors develop long-term cognitive complications post-discharge. The present study identifies a novel regulatory relationship between amyloid-ß (Aß) and the inflammasome-caspase-1 axis as key innate immune mediators that define sepsis outcomes. Methods: Medical ICU patients and healthy individuals were consented for blood and clinical data collection. Plasma cytokine, caspase-1 and Aß levels were measured. Data were compared against indices of multiorgan injury and other clinical parameters. Additionally, recombinant proteins were tested in vitro to examine the effect of caspase-1 on a functional hallmark of Aß, namely aggregation. Results: Plasma caspase-1 levels displayed the best predictive value in discriminating ICU patients with sepsis from non-infected ICU patients (area under the receiver operating characteristic curve=0.7080). Plasma caspase-1 and the Aß isoform Aßx-40 showed a significant positive correlation and Aßx-40 associated with organ injury. Additionally, Aß plasma levels continued to rise from time of ICU admission to 7 days post-admission. In silico, Aß harbours a predicted caspase-1 cleavage site, and in vitro studies demonstrated that caspase-1 cleaved Aß to inhibit its auto-aggregation, suggesting a novel regulatory relationship. Conclusions: Aßx-40 and caspase-1 are potentially useful early indicators of sepsis and its attendant organ injury. Additionally, Aßx-40 has emerged as a potential culprit in the ensuing development of post-ICU syndrome.

11.
JCI Insight ; 9(5)2024 Mar 08.
Article in English | MEDLINE | ID: mdl-38290089

ABSTRACT

Rare diseases are underrepresented in biomedical research, leading to insufficient awareness. Zhu-Tokita-Takenouchi-Kim (ZTTK) syndrome is a rare disease caused by genetic alterations that result in heterozygous loss of function of SON. While patients with ZTTK syndrome live with numerous symptoms, the lack of model organisms hampers our understanding of SON and this complex syndrome. Here, we developed Son haploinsufficiency (Son+/-) mice as a model of ZTTK syndrome and identified the indispensable roles of Son in organ development and hematopoiesis. Son+/- mice recapitulated clinical symptoms of ZTTK syndrome, including growth retardation, cognitive impairment, skeletal abnormalities, and kidney agenesis. Furthermore, we identified hematopoietic abnormalities in Son+/- mice, including leukopenia and immunoglobulin deficiency, similar to those observed in human patients. Surface marker analyses and single-cell transcriptome profiling of hematopoietic stem and progenitor cells revealed that Son haploinsufficiency shifted cell fate more toward the myeloid lineage but compromised lymphoid lineage development by reducing genes required for lymphoid and B cell lineage specification. Additionally, Son haploinsufficiency caused inappropriate activation of erythroid genes and impaired erythropoiesis. These findings highlight the importance of the full gene expression of Son in multiple organs. Our model serves as an invaluable research tool for this rare disease and related disorders associated with SON dysfunction.


Subject(s)
Hematopoiesis , Rare Diseases , Animals , Humans , Mice , Gene Expression Profiling , Hematopoiesis/genetics , Mutation
12.
Am Surg ; 89(11): 4536-4541, 2023 Nov.
Article in English | MEDLINE | ID: mdl-35979859

ABSTRACT

INTRODUCTION: Studies have demonstrated that trauma patients with early-ventilator associated pneumonia (early-VAP, < 7 days) have decreased risk of methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa infections. We hypothesize that routinely using broad-spectrum antibiotics is unnecessary to treat trauma patients with the diagnosis of early-VAP. METHODS: This retrospective cohort study included adult trauma patients with the diagnosis of VAP. The primary outcome was the presence of MRSA and/or P. aeruginosa in patients with early- and late-VAP. Secondary outcomes included the bacterial susceptibility of pathogens to methicillin, ampicillin/sulbactam, ceftriaxone, piperacillin/tazobactam, and cefepime. Intensive care unit (ICU) and hospital length of stay (LOS), ventilator-free days, and in-hospital mortality were also collected. RESULTS: 164 patients met inclusion criteria, and 208 organisms (n = 90 early vs n = 118 late) were identified by respiratory culture. The incidence of MRSA and P. aeruginosa in early-VAP was 7.7% (7/90) and 5.6% (5/90), respectively. The susceptibility of bacteria causing early-VAP to ampicillin/sulbactam and ceftriaxone was 73.3% (66/90) and 83.3% (75/90), respectively. Ventilator-free days at 30 days was similar between groups (P = .649). Patients with late-VAP spent more time in the ICU (P = .040); however, in-hospital mortality was higher in the early-VAP group (P = .012). CONCLUSIONS: Ampicillin/sulbactam or ceftriaxone monotherapy did not provide reliable broad-spectrum coverage for early-VAP in our cohort. These findings highlight the importance of each institution performing a similar analysis to ensure adequate initial treatment of VAP.


Subject(s)
Methicillin-Resistant Staphylococcus aureus , Pneumonia, Ventilator-Associated , Adult , Humans , Pneumonia, Ventilator-Associated/drug therapy , Pneumonia, Ventilator-Associated/diagnosis , Sulbactam/therapeutic use , Retrospective Studies , Ceftriaxone/therapeutic use , Anti-Bacterial Agents/therapeutic use , Ampicillin/therapeutic use , Bacteria , Intensive Care Units
13.
bioRxiv ; 2023 Nov 19.
Article in English | MEDLINE | ID: mdl-38014320

ABSTRACT

Rare diseases are underrepresented in biomedical research, leading to insufficient awareness. Zhu-Tokita-Takenouchi-Kim (ZTTK) syndrome is a rare disease caused by genetic alterations that result in heterozygous loss-of-function of SON. While ZTTK syndrome patients suffer from numerous symptoms, the lack of model organisms hamper our understanding of both SON and this complex syndrome. Here, we developed Son haploinsufficiency (Son+/-) mice as a model of ZTTK syndrome and identified the indispensable roles of Son in organ development and hematopoiesis. Son+/- mice recapitulated clinical symptoms of ZTTK syndrome, including growth retardation, cognitive impairment, skeletal abnormalities, and kidney agenesis. Furthermore, we identified hematopoietic abnormalities in Son+/- mice, similar to those observed in human patients. Surface marker analyses and single-cell transcriptome profiling of hematopoietic stem and progenitor cells revealed that Son haploinsufficiency inclines cell fate toward the myeloid lineage but compromises lymphoid lineage development by reducing key genes required for lymphoid and B cell lineage specification. Additionally, Son haploinsufficiency causes inappropriate activation of erythroid genes and impaired erythroid maturation. These findings highlight the importance of the full gene dosage of Son in organ development and hematopoiesis. Our model serves as an invaluable research tool for this rare disease and related disorders associated with SON dysfunction.

14.
Proc Natl Acad Sci U S A ; 106(34): 14490-5, 2009 Aug 25.
Article in English | MEDLINE | ID: mdl-19706534

ABSTRACT

Complement receptors (CRs) CD21 and CD35 form a coreceptor with CD19 and CD81 on murine B cells that when coligated with the B-cell receptor lowers the threshold of activation by several orders of magnitude. This intrinsic signaling role is thought to explain the impaired humoral immunity of mice bearing deficiency in CRs. However, CRs have additional roles on B cells independent of CD19, such as transport of C3-coated immune complexes and regulation of C4 and C3 convertase. To test whether association of CR with CD19 is necessary for their intrinsic activation-enhancing role, knockin mice expressing mutant receptors, Cr2(Delta/Deltagfp), that bind C3 ligands but do not signal through CD19 were constructed. We found that uncoupling of CR and CD19 significantly diminishes survival of germinal center B cells and secondary antibody titers. However, B memory is less impaired relative to mice bearing a complete deficiency in CRs on B cells. These findings confirm the importance of interaction of CR and CD19 for coreceptor activity in humoral immunity but identify a role for CR in B-cell memory independent of CD19.


Subject(s)
Antigens, CD19/metabolism , B-Lymphocytes/metabolism , Receptors, Complement 3d/metabolism , Animals , Antibody Formation/immunology , Antigens, CD19/genetics , B-Lymphocytes/cytology , B-Lymphocytes/immunology , Bone Marrow/immunology , Bone Marrow/metabolism , Bone Marrow Transplantation , Flow Cytometry , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Haptens , Hemocyanins/immunology , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Immunization , Immunologic Memory/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , Mutation , Receptors, Complement 3b/genetics , Receptors, Complement 3b/metabolism , Receptors, Complement 3d/genetics , Spleen/immunology , Spleen/metabolism
15.
Toxins (Basel) ; 14(2)2022 02 18.
Article in English | MEDLINE | ID: mdl-35202178

ABSTRACT

The Gram-negative, opportunistic pathogen Pseudomonas aeruginosa utilizes a type III secretion system to inject exoenzyme effectors into a target host cell. Of the four best-studied exoenzymes, ExoU causes rapid cell damage and death. ExoU is a phospholipase A2 (PLA2) that hydrolyses host cell membranes, and P. aeruginosa strains expressing ExoU are associated with poor outcomes in critically ill patients with pneumonia. While the effects of ExoU on lung epithelial and immune cells are well studied, a role for ExoU in disrupting lung endothelial cell function has only recently emerged. Lung endothelial cells maintain a barrier to fluid and protein flux into tissue and airspaces and regulate inflammation. Herein, we describe a pulmonary microvascular endothelial cell (PMVEC) culture infection model to examine the effects of ExoU. Using characterized P. aeruginosa strains and primary clinical isolates, we show that strains expressing ExoU disrupt PMVEC barrier function by causing substantial PMVEC damage and lysis, in a PLA2-dependent manner. In addition, we show that strains expressing ExoU activate the pro-inflammatory caspase-1, in a PLA2-dependent manner. Considering the important roles for mitochondria and oxidative stress in regulating inflammatory responses, we next examined the effects of ExoU on reactive oxygen species production. Infection of PMVECs with P. aeruginosa strains expressing ExoU triggered a robust oxidative stress compared to strains expressing other exoenzyme effectors. We also provide evidence that, intriguingly, ExoU PLA2 activity was detectable in mitochondria and mitochondria-associated membrane fractions isolated from P. aeruginosa-infected PMVECs. Interestingly, ExoU-mediated activation of caspase-1 was partially inhibited by reactive oxygen species scavengers. Together, these data suggest ExoU exerts pleiotropic effects on PMVEC function during P. aeruginosa infection that may inhibit endothelial barrier and inflammatory functions.


Subject(s)
Bacterial Proteins/toxicity , Caspase 1/drug effects , Cell Death/drug effects , Chemical and Drug Induced Liver Injury/physiopathology , Endothelial Cells/drug effects , Pseudomonas Infections/physiopathology , Pseudomonas aeruginosa/genetics , Caspase 1/metabolism , Genetic Variation , Genotype , Humans , Inflammation/chemically induced , Inflammation/physiopathology , Pseudomonas Infections/genetics
16.
Vaccines (Basel) ; 9(8)2021 Aug 12.
Article in English | MEDLINE | ID: mdl-34452019

ABSTRACT

AIM: To determine the relationship between gene expression profile (GEP) and overall survival (OS) by NanoString following treatment with Vigil. PATIENTS AND METHODS: Recurrent ovarian cancer patients (n = 21) enrolled in prior clinical trials. RESULTS: GEP stratified by TISHIGH vs. TISLOW demonstrated OS benefit (NR vs. 5.8 months HR 0.23; p = 0.0379), and in particular, MHC-II elevated baseline expression was correlated with OS advantage (p = 0.038). Moreover, 1-year OS was 75% in TISHIGH patients vs. 25% in TISLOW (p = 0.03795). OS was also correlated with positive γ-IFN ELISPOT response, 36.8 vs. 23.0 months (HR 0.19, p = 0.0098). CONCLUSION: Vigil demonstrates OS benefit in correlation with TISHIGH score, elevated MHC-II expression and positive γ-IFN ELISPOT in recurrent ovarian cancer patients.

17.
J Exp Med ; 196(9): 1189-99, 2002 Nov 04.
Article in English | MEDLINE | ID: mdl-12417629

ABSTRACT

To dissect the influence of CD21/CD35 and FcgammaRIIB in antigen retention and humoral memory, we used an adoptive transfer model in which antigen-primed B and T lymphocytes were given to sublethally irradiated wild-type mice or mice deficient in CD21/CD35 (Cr2(-/-)) or FcgammaRIIB receptors (FcgammaRIIB(-/-)). Cr2(-/-) chimeras showed impaired memory as characterized by a decrease in antibody titer, reduced frequency of antibody secreting cells, an absence of affinity maturation, and significantly reduced recall response. The impaired memory in Cr2(-/-) chimeras corresponded with the reduced frequency of antigen-specific memory B cells. Interestingly, FcgammaRIIB(-/-) chimeras showed a differential phenotype with impaired splenic but normal bone marrow responses. These data suggest that CD21/CD35 on stroma, including follicular dendritic cells, is critical to the maintenance of long-term B lymphocyte memory.


Subject(s)
Antigens, CD/immunology , B-Lymphocytes/immunology , Immunologic Memory/immunology , Receptors, Complement 3b/immunology , Receptors, Complement 3d/immunology , Receptors, IgG/immunology , Stromal Cells/immunology , Animals , Antibodies/blood , Haptens , Hemocyanins/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , Time Factors
18.
Article in English | MEDLINE | ID: mdl-32405439

ABSTRACT

BACKGROUND: The pathological consequences of interaction between environmental carbon pollutants and microbial antigens have not been fully explored. We developed a murine model of multi-wall carbon nanotube (MWCNT)-elicited granulomatous disease which bears a striking resemblance to sarcoidosis, a human granulomatous disease. Because of reports describing lymphocyte reactivity to mycobacterial antigens in sarcoidosis patients, we hypothesized that addition of mycobacterial antigen (ESAT-6) to MWCNT might elicit activation in T cells. METHODS: Macrophage-specific peroxisome-proliferator-activated receptor gamma (PPARγ) knock out (KO) mice were studied along with wild-type mice because our previous report indicated PPARγ deficiency in sarcoidosis alveolar macrophages. MWCNT+ESAT-6 were instilled into mice. Controls received vehicle (surfactant-PBS) or ESAT-6 and were evaluated 60 days post-instillation. As noted in our recent publication, lung tissues from PPARγ KO mice instilled with MWCNT+ESAT-6 yielded more intensive pathophysiology, with elevated fibrosis. RESULTS: Inspection of mediastinal lymph nodes (MLN) revealed no granulomas but deposition of MWCNT. MLN cell counts were higher in PPARγ KO than in wild-type instilled with MWCNT+ESAT-6. Moreover, the CD4:CD8 T cell ratio, a major clinical metric for human disease, was increased in PPARγ KO mice. Bronchoalveolar lavage (BAL) cells from PPARγ KO mice instilled with MWCNT+ESAT-6 displayed increased Th17 cell markers (RORγt, IL-17A, CCR6) which associate with elevated fibrosis. CONCLUSION: These findings suggest that PPARγ deficiency in macrophages may promote ESAT-6-associated T cell activation in the lung, and that the MWCNT+ESAT-6 model may offer new insights into pathways of lymphocyte-mediated sarcoidosis histopathology.

19.
Invest Ophthalmol Vis Sci ; 60(12): 3952-3962, 2019 09 03.
Article in English | MEDLINE | ID: mdl-31560369

ABSTRACT

Purpose: γδ T cells offer an important early immune defense against many different pathogens, both bacterial and viral. Herein, we examined the capacity of γδ T cell subsets to provide protection in the cornea against herpes simplex virus-1 (HSV-1). Methods: C57Bl/6 (wild-type [WT]), γδ T-cell deficient (TCRδ-/-) and CCR6-deficient (CCR6-/-) mice were infected intracorneally with HSV-1. At multiple time points following infection, corneas were excised, and cells were immunostained for surface markers, intracellular cytokines, and analyzed using flow cytometry. WT and CCR6-/- γδ T cells were adoptively transferred into TCRδ-/- mice and corneal scores and survival were measured. Results: Intracorneal infection of mice lacking γδ T cells exhibited increased corneal opacity scores, elevated viral titers, and higher mortality compared with WT mice. Both CCR6+ and CCR6neg γδ T cell subsets were observed in corneas after virus infection. CCR6+ γδ T cells produced IL-17A and were predominantly CD44+CD62L+, consistent with natural IL-17+ γδ T cells. In contrast IL-17A production by CCR6neg γδ T cells was infrequent, and this subset was largely single positive for CD62L or CD44. The CCR6+ subset appeared to provide protection against HSV-1 as follows: (1) CCR6-/- mice had more severe corneal opacity compared with WT mice; and (2) adoptive transfer of γδ T cells from WT mice restored protection in TCRδ-/- mice whereas transfer of γδ T cells from CCR6-/- mice did not. Conclusions: γδ T cells in the cornea can be divided into CCR6+ and CCR6neg subsets with the former conferring protection early after intracorneal HSV-1 infection.


Subject(s)
Corneal Opacity/prevention & control , Herpesvirus 1, Human/physiology , Intraepithelial Lymphocytes/immunology , Keratitis, Herpetic/prevention & control , Receptors, CCR6/immunology , Adoptive Transfer , Animals , Cornea/virology , Corneal Opacity/immunology , Corneal Opacity/virology , Cytokines/metabolism , Enzyme-Linked Immunosorbent Assay , Female , Flow Cytometry , Interleukin-17/metabolism , Keratitis, Herpetic/immunology , Keratitis, Herpetic/virology , Killer Cells, Natural/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , Trigeminal Ganglion/virology , Viral Plaque Assay
20.
Curr Dir Autoimmun ; 7: 4-32, 2004.
Article in English | MEDLINE | ID: mdl-14719373

ABSTRACT

Early studies, largely based on in vitro models, revealed potential functional roles for the components of the CD19/21/81 complex in B cell proliferation and antibody production. These studies also identified signal transduction pathways linked to these receptors. Over the last decade, studies on knockout mice defined the biologic functions of CD19, CD21 and CD81. This review focuses on current attempts to use these receptors as tools to understand how the immune system regulates responsiveness and tolerance, while correlating specific biochemical pathways with biologic function.


Subject(s)
Antigens, CD19/physiology , B-Lymphocytes/physiology , Receptors, Complement 3d/physiology , Signal Transduction/physiology , Animals , Antibody Formation , Antigen Presentation , Calcium/metabolism , Germinal Center/physiology , Humans , Lymphocyte Activation , Membrane Microdomains/physiology , Phosphorylation
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