ABSTRACT
Chronic bone loss is an under-recognized complication of malaria, the underlying mechanism of which remains incompletely understood. We have previously shown that persistent accumulation of Plasmodium products in the bone marrow leads to chronic inflammation in osteoblast (OB) and osteoclast (OC) precursors causing bone loss through MyD88, an adaptor molecule for diverse inflammatory signals. However, the specific contribution of MyD88 signaling in OB or OC precursors in malaria-induced bone loss remains elusive. To assess the direct cell-intrinsic role of MyD88 signaling in adult bone metabolism under physiological and infection conditions, we used the Lox-Cre system to specifically deplete MyD88 in the OB or OC lineages. Mice lacking MyD88 primarily in the maturing OBs showed a comparable decrease in trabecular bone density by microcomputed tomography to that of controls after Plasmodium yoelii non-lethal infection. In contrast, mice lacking MyD88 in OC precursors showed significantly less trabecular bone loss than controls, suggesting that malaria-mediated inflammatory mediators are primarily controlled by MyD88 in the OC lineage. Surprisingly, however, depletion of MyD88 in OB, but not in OC, precursors resulted in reduced bone mass with decreased bone formation rates in the trabecular areas of femurs under physiological conditions. Notably, insulin-like growth factor-1, a key molecule for OB differentiation, was significantly lower locally and systemically when MyD88 was depleted in OBs. Thus, our data demonstrate an indispensable intrinsic role for MyD88 signaling in OB differentiation and bone formation, while MyD88 signaling in OC lineages plays a partial role in controlling malaria-induced inflammatory mediators and following bone pathology. These findings may lead to the identification of novel targets for specific intervention of bone pathologies, particularly in malaria-endemic regions.
Subject(s)
Bone Remodeling , Homeostasis , Malaria , Mice, Knockout , Myeloid Differentiation Factor 88 , Osteoblasts , Osteoclasts , Animals , Myeloid Differentiation Factor 88/metabolism , Myeloid Differentiation Factor 88/genetics , Malaria/immunology , Osteoblasts/metabolism , Osteoblasts/immunology , Mice , Osteoclasts/metabolism , Osteoclasts/immunology , Plasmodium yoelii/immunology , Mice, Inbred C57BLABSTRACT
5,6-dimethylxanthenone-4-acetic acid (DMXAA) is a mouse-selective stimulator of interferon gene (STING) agonist exerting STING-dependent anti-tumor activity. Although DMXAA cannot fully activate human STING, DMXAA reached phase III in lung cancer clinical trials. How DMXAA is effective against human lung cancer is completely unknown. Here, we show that DMXAA is a partial STING agonist interfering with agonistic STING activation, which may explain its partial anti-tumor effect observed in humans, as STING was reported to be pro-tumorigenic for lung cancer cells with low antigenicity. Furthermore, we developed a DMXAA derivative-3-hydroxy-5-(4-hydroxybenzyl)-4-methyl-9H-xanthen-9-one (HHMX)-that can potently antagonize STING-mediated immune responses both in humans and mice. Notably, HHMX suppressed aberrant responses induced by STING gain-of-function mutations causing STING-associated vasculopathy with onset in infancy (SAVI) in in vitro experiments. Furthermore, HHMX treatment suppressed aberrant STING pathway activity in peripheral blood mononuclear cells from SAVI patients. Lastly, HHMX showed a potent therapeutic effect in SAVI mouse model by mitigating disease progression. Thus, HHMX offers therapeutic potential for STING-associated autoinflammatory diseases.
Subject(s)
Lung Neoplasms , Membrane Proteins , Xanthones , Humans , Mice , Animals , Membrane Proteins/metabolism , Leukocytes, Mononuclear/metabolism , Lung/metabolismABSTRACT
Bone marrow is a dynamic organ composed of stem cells that constantly receive signals from stromal cells and other hematopoietic cells in the niches of the bone marrow to maintain hematopoiesis and generate immune cells. Perturbation of the bone marrow microenvironment by infection and inflammation affects hematopoiesis and may affect immune cell development. Little is known about the effect of malaria on the bone marrow stromal cells that govern the hematopoietic stem cell (HSC) niche. In this study, we demonstrate that the mesenchymal stromal CXCL12-abundant reticular (CAR) cell population is reduced during acute malaria infection. The reduction of CXCL12 and interleukin-7 signals in the bone marrow impairs the lymphopoietic niche, leading to the depletion of common lymphoid progenitors, B cell progenitors, and mature B cells, including plasma cells in the bone marrow. We found that interferon-γ (IFNγ) is responsible for the upregulation of Sca1 on CAR cells, yet the decline in CAR cell and B cell populations in the bone marrow is IFNγ-independent. In contrast to the decline in B cell populations, HSCs and multipotent progenitors increased with the expansion of myelopoiesis and erythropoiesis, indicating a bias in the differentiation of multipotent progenitors during malaria infection. These findings suggest that malaria may affect host immunity by modulating the bone marrow niche.
Subject(s)
B-Lymphocytes , Bone Marrow , Chemokine CXCL12 , Malaria , Mice, Inbred C57BL , Animals , Chemokine CXCL12/metabolism , Chemokine CXCL12/immunology , Mice , Malaria/immunology , Malaria/parasitology , B-Lymphocytes/immunology , Bone Marrow/immunology , Bone Marrow/parasitology , Stem Cell Niche/immunology , Interferon-gamma/metabolism , Interferon-gamma/immunology , Hematopoietic Stem Cells/immunology , Hematopoietic Stem Cells/metabolismABSTRACT
Purpose: Breast cancers exhibit molecular heterogeneity, leading to diverse clinical outcomes and therapeutic responses. Immune checkpoint inhibitors targeting PD-L1 have shown promise in various malignancies, including breast cancer. Lipocalin 2 (LCN2) has also been associated with tumor aggressiveness and prognostic potential in breast cancers. However, the expression of PD-L1 and LCN2 in breast cancer subtypes and their prognostic implications remains poorly investigated. Methods: A retrospective analysis of 89 primary breast cancer cases was conducted to assess PD-L1 and LCN2 expressions using immunohistochemistry. Cases were classified into four different molecular subtypes based on ER, PR, HER2, and Ki-67 status. Associations between PD-L1 and LCN2 expressions and various prognostic factors were examined. Results: Although low expression of LCN2 (Allred score of <3) was observed even in normal breast tissue, LCN2 expression with increasing Allred score (≥3) positively correlated with the histological grade, high Ki-67 proliferation index, and ER/PR negativity. Significant elevations of LCN2 and PD-L1 expressions were observed in triple-negative and HER2-positive breast cancers. Conclusion: The results of the study highlight the association of LCN2 with known prognostic factors and molecular subtypes. To identify potential immunotherapy recipients, it would be useful to evaluate LCN2 as well as PD-L1 immune targets in different subgroups of breast cancer patients. Further studies with larger patient numbers are warranted to validate these observations and establish standardized scoring criteria for LCN2 expression assessment.
ABSTRACT
The Plasmodium life cycle involves differentiation into multiple morphologically distinct forms, a process regulated by developmental stage-specific gene expression. Histone proteins are involved in epigenetic regulation in eukaryotes, and the histone variant H3.3 plays a key role in the regulation of gene expression and maintenance of genomic integrity during embryonic development in mice. However, the function of H3.3 through multiple developmental stages in Plasmodium remains unknown. To examine the function of H3.3, h3.3-deficient mutants (Δh3.3) were generated in P. berghei. The deletion of h3.3 was not lethal in blood stage parasites, although it had a minor effect of the growth rate in blood stage; however, the in vitro ookinete conversion rate was significantly reduced, and the production of the degenerated form was increased. Regarding the mosquito stage development of Δh3.3, oocysts number was significantly reduced, and no sporozoite production was observed. The h3.3 gene complemented mutant have normal development in mosquito stage producing mature oocysts and salivary glands contained sporozoites, and interestingly, the majority of H3.3 protein was detected in female gametocytes. However, Δh3.3 male and female gametocyte production levels were comparable to the wild-type levels. Transcriptome analysis of Δh3.3 male and female gametocytes revealed the upregulation of several male-specific genes in female gametocytes, suggesting that H3.3 functions as a transcription repressor of male-specific genes to maintain sexual identity in female gametocytes. This study provides new insights into the molecular biology of histone variants H3.3 which plays a critical role on zygote-to-oocyst development in primitive unicellular eukaryotes.
Subject(s)
Histones , Malaria , Parasites , Plasmodium berghei , Plasmodium , Animals , Female , Male , Mice , Epigenesis, Genetic , Histones/genetics , Malaria/parasitology , Oocysts , Plasmodium berghei/genetics , Plasmodium berghei/physiology , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , Sporozoites/physiology , Zygote/metabolismABSTRACT
Gene editing using engineered nucleases frequently produces unintended genetic lesions in hematopoietic stem cells (HSCs). Gene-edited HSC cultures thus contain heterogeneous populations, the majority of which either do not carry the desired edit or harbor unwanted mutations. In consequence, transplanting edited HSCs carries the risks of suboptimal efficiency and of unwanted mutations in the graft. Here, we present an approach for expanding gene-edited HSCs at clonal density, allowing for genetic profiling of individual clones before transplantation. We achieved this by developing a defined, polymer-based expansion system and identifying long-term expanding clones within the CD201+CD150+CD48-c-Kit+Sca-1+Lin- population of precultured HSCs. Using the Prkdcscid immunodeficiency model, we demonstrate that we can expand and profile edited HSC clones to check for desired and unintended modifications, including large deletions. Transplantation of Prkdc-corrected HSCs rescued the immunodeficient phenotype. Our ex vivo manipulation platform establishes a paradigm to control genetic heterogeneity in HSC gene editing and therapy.
Subject(s)
Gene Editing , Hematopoietic Stem Cell Transplantation , Genetic Heterogeneity , Hematopoietic Stem Cells , Phenotype , Clone CellsABSTRACT
In women with unexplained infertility (UI) and recurrent in vitro fertilization (IVF) failures, the etiology is often unclear. Endometrial immune perturbations and the use of immune markers associated with these dysregulations are of great interest in the diagnosis and treatment of UI. However, reliable biomarkers and standardized quantification methods are lacking. Here, to address endometrial immune dysregulation in UI patients with recurrent IVF failures, we performed endometrial tissue sampling and immunostaining of CD56 (uNK), CD138, and BCL-6. Of these cases, 57.9% had positive CD56 in the endometrial stroma, while 46.1% had positive BCL-6 in the glandular epithelium, and 14.5% of the cases were found to be positive for CD138. Combined staining rates were 60.5%, 68.4%, and 71.05% for (CD56 or BCL-6), (CD56 or CD138), and (CD56, BCL-6, or CD138), respectively. There was a significant correlation between CD56 and BCL-6 positivity, while CD138 positivity was an independent parameter. After the recommended targeted therapy, pregnancy rates were found to increase from 58.5% to 61.6% and 73.8% in CD56-positive, (CD56- or BCL-6-positive), and (CD56-, BCL-6-, or CD138-positive) cases, respectively. Notably, a retrospective evaluation of digital pathology and light microscopy results showed a significant correlation. This study suggests that the examination of CD56, BCL-6, and CD138 in the same endometrial sample may be an effective method in determining the etiology of UI and reaching an early diagnosis and treatment options. Moreover, digital pathology can be used in the evaluation of CD56 and BCL-6 to provide objective, rapid, and reliable results.
ABSTRACT
Adjuvants are important vaccine components, composed of a variety of chemical and biological materials that enhance the vaccine antigen-specific immune responses by stimulating the innate immune cells in both direct and indirect manners to produce a variety cytokines, chemokines, and growth factors. It has been developed by empirical methods for decades and considered difficult to choose a single screening method for an ideal vaccine adjuvant, due to their diverse biochemical characteristics, complex mechanisms of, and species specificity for their adjuvanticity. We therefore established a robust adjuvant screening strategy by combining multiparametric analysis of adjuvanticity in vivo and immunological profiles in vitro (such as cytokines, chemokines, and growth factor secretion) of various library compounds derived from hot-water extracts of herbal medicines, together with their diverse distribution of nano-sized physical particle properties with a machine learning algorithm. By combining multiparametric analysis with a machine learning algorithm such as rCCA, sparse-PLS, and DIABLO, we identified that human G-CSF and mouse RANTES, produced upon adjuvant stimulation in vitro, are the most robust biological parameters that can predict the adjuvanticity of various library compounds. Notably, we revealed a certain nano-sized particle population that functioned as an independent negative parameter to adjuvanticity. Finally, we proved that the two-step strategy pairing the negative and positive parameters significantly improved the efficacy of screening and a screening strategy applying principal component analysis using the identified parameters. These novel parameters we identified for adjuvant screening by machine learning with multiple biological and physical parameters may provide new insights into the future development of effective and safe adjuvants for human use.
Subject(s)
Adjuvants, Vaccine , Vaccines , Adjuvants, Immunologic/chemistry , Adjuvants, Immunologic/pharmacology , Adjuvants, Pharmaceutic , Animals , Cytokines , Herbal Medicine , Machine Learning , MiceABSTRACT
Agonists for TLR9 and stimulator of IFN genes (STING) offer therapeutic applications as both anti-tumor agents and vaccine adjuvants, though their clinical applications are limited; the clinically available TLR9 agonist is a weak IFN inducer and STING agonists induce undesired type 2 immunity. Yet, combining TLR9 and STING agonists overcame these limitations by synergistically inducing innate and adaptive IFNγ to become an advantageous type 1 adjuvant, suppressing type 2 immunity, in addition to exerting robust anti-tumor activities when used as a monotherapeutic agent for cancer immunotherapy. Here, we sought to decipher the immunological mechanisms behind the synergism mediated by TLR9 and STING agonists and found that their potent anti-tumor immunity in a Pan02 peritoneal dissemination model of pancreatic cancer was achieved only when agonists for TLR9 and STING were administered locally, and was via mechanisms involving CD4 and CD8 T cells as well as the co-operative action of IL-12 and type I IFNs. Rechallenge studies of long-term cancer survivors suggested that the elicitation of Pan02-specific memory responses provides protection against the secondary tumor challenge. Mechanistically, we found that TLR9 and STING agonists synergistically induce IL-12 and type I IFN production in murine APCs. The synergistic effect of the TLR9 and STING agonists on IL-12p40 was at protein, mRNA and promoter activation levels, and transcriptional regulation was mediated by a 200 bp region situated 983 bp upstream of the IL-12p40 transcription initiation site. Such intracellular transcriptional synergy may hold a key in successful cancer immunotherapy and provide further insights into dual agonism of innate immune sensors during host homeostasis and diseases.
Subject(s)
Membrane Proteins , Neoplasms , Toll-Like Receptor 9 , Adjuvants, Immunologic/pharmacology , Animals , Immunotherapy , Interleukin-12 , Interleukin-12 Subunit p40 , Membrane Proteins/metabolism , Mice , Toll-Like Receptor 9/metabolismABSTRACT
The germinal center (GC) is a site where somatic hypermutation and clonal selection are coupled for antibody affinity maturation against infections. However, how GCs are formed and regulated is incompletely understood. Here, we identified an unexpected role of Tank-binding kinase-1 (TBK1) as a crucial B cell-intrinsic factor for GC formation. Using immunization and malaria infection models, we show that TBK1-deficient B cells failed to form GC despite normal Tfh cell differentiation, although some malaria-infected B cell-specific TBK1-deficient mice could survive by GC-independent mechanisms. Mechanistically, TBK1 phosphorylation elevates in B cells during GC differentiation and regulates the balance of IRF4/BCL6 expression by limiting CD40 and BCR activation through noncanonical NF-κB and AKTT308 signaling. In the absence of TBK1, CD40 and BCR signaling synergistically enhanced IRF4 expression in Pre-GC, leading to BCL6 suppression, and therefore failed to form GCs. As a result, memory B cells generated from TBK1-deficient B cells fail to confer sterile immunity upon reinfection, suggesting that TBK1 determines B cell fate to promote long-lasting humoral immunity.
Subject(s)
B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Germinal Center/immunology , Germinal Center/metabolism , Host-Pathogen Interactions , Infections/etiology , Protein Serine-Threonine Kinases/genetics , Animals , Biomarkers , CD40 Antigens/metabolism , Gene Expression Regulation , Gene Knockdown Techniques , Host-Pathogen Interactions/genetics , Host-Pathogen Interactions/immunology , Immunity, Humoral , Immunization , Mice , Protein Serine-Threonine Kinases/metabolism , Receptors, Antigen, B-Cell/metabolism , Signal Transduction , T-Lymphocytes, Helper-Inducer/immunology , T-Lymphocytes, Helper-Inducer/metabolismABSTRACT
NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasome activation is beneficial during infection and vaccination but, when uncontrolled, is detrimental and contributes to inflammation-driven pathologies. Hence, discovering endogenous mechanisms that regulate NLRP3 activation is important for disease interventions. Activation of NLRP3 is regulated at the transcriptional level and by posttranslational modifications. Here, we describe a posttranslational phospho-switch that licenses NLRP3 activation in macrophages. The ON switch is controlled by the protein phosphatase 2A (PP2A) downstream of a variety of NLRP3 activators in vitro and in lipopolysaccharide-induced peritonitis in vivo. The OFF switch is regulated by two closely related kinases, TANK-binding kinase 1 (TBK1) and I-kappa-B kinase epsilon (IKKε). Pharmacological inhibition of TBK1 and IKKε, as well as simultaneous deletion of TBK1 and IKKε, but not of either kinase alone, increases NLRP3 activation. In addition, TBK1/IKKε inhibitors counteract the effects of PP2A inhibition on inflammasome activity. We find that, mechanistically, TBK1 interacts with NLRP3 and controls the pathway activity at a site distinct from NLRP3-serine 3, previously reported to be under PP2A control. Mutagenesis of NLRP3 confirms serine 3 as an important phospho-switch site but, surprisingly, reveals that this is not the sole site regulated by either TBK1/IKKε or PP2A, because all retain the control over the NLRP3 pathway even when serine 3 is mutated. Altogether, a model emerges whereby TLR-activated TBK1 and IKKε act like a "parking brake" for NLRP3 activation at the time of priming, while PP2A helps remove this parking brake in the presence of NLRP3 activating signals, such as bacterial pore-forming toxins or endogenous danger signals.
Subject(s)
I-kappa B Kinase/genetics , Inflammasomes/genetics , NLR Family, Pyrin Domain-Containing 3 Protein/genetics , Protein Serine-Threonine Kinases/genetics , Signal Transduction/genetics , Animals , Cell Line , Female , Humans , Macrophages/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Phosphorylation/geneticsABSTRACT
Cerebral malaria (CM) is a life-threatening complication of the malaria disease caused by Plasmodium falciparum infection and is responsible for the death of half a million people annually. The molecular pathogenesis underlying CM in humans is not completely understood, although sequestration of infected erythrocytes in cerebral microvessels is thought to play a major role. In contrast, experimental cerebral malaria (ECM) models in mice have been thought to be distinct from human CM, and are mainly caused by inflammatory mediators. Here, to understand the spatial distribution and the potential sequestration of parasites in the whole-brain microvessels during a mouse model of ECM, we utilized the new tissue-clearing method CUBIC (Clear, Unobstructed, Brain/Body Imaging Cocktails and Computational analysis) with light-sheet fluorescent microscopy (LSFM), and reconstructed images in three dimensions (3D). We demonstrated significantly greater accumulation of Plasmodium berghei ANKA (PbANKA) parasites in the olfactory bulb (OB) of mice, compared with the other parts of the brain, including the cerebral cortex, cerebellum and brainstem. Furthermore, we show that PbANKA parasites preferentially accumulate in the brainstem when the OB is surgically removed. This study therefore not only highlights a successful application of CUBIC tissue-clearing technology to visualize the whole brain and its microvessels during ECM, but it also shows CUBIC's future potential for visualizing pathological events in the whole ECM brain at the cellular level, an achievement that would greatly advance our understanding of human cerebral malaria.
Subject(s)
Brain/pathology , Malaria, Cerebral/pathology , Animals , Brain/immunology , Brain/parasitology , Disease Models, Animal , Malaria, Cerebral/immunology , Malaria, Cerebral/parasitology , Mice , Mice, Inbred C57BL , Plasmodium berghei/immunologyABSTRACT
Due to the rapid onset and spread of the COVID-19 pandemic, the treatment of COVID-19 patients by hydroxychloroquine alone or in combination with other drugs has captured a great deal of attention and triggered considerable debate. Historically, the worldwide use of quinoline based-drugs has led to a spectacular reduction in death from malaria. Unfortunately, scientists have been forced to seek alternative drugs to treat malaria due to the emergence of chloroquine-resistant parasites in the 1960s. The repurposing of hydroxychloroquine against viral infections, various types of cancer and autoimmune diseases has been ongoing for more than 70 years, with no clear understanding of its mechanism of action (MOA). Here, we closely examine the MOA of this old but influential drug in and beyond malaria. Better insights into how chloroquine targets the host's cellular and immune responses may help to develop applications against to new pathogens and diseases, and perhaps even restore the clinical utility of chloroquine against malaria.
Subject(s)
Chloroquine/pharmacology , Chloroquine/therapeutic use , Malaria/drug therapy , Animals , Humans , Hydroxychloroquine/pharmacology , Hydroxychloroquine/therapeutic use , Pandemics/prevention & control , SARS-CoV-2/drug effects , COVID-19 Drug TreatmentABSTRACT
Heparin is used extensively as an anticoagulant in a broad range of diseases and procedures; however, its biological effects are not limited to coagulation and remain incompletely understood. Heparin usage can lead to the life-threatening complication known as heparin-induced thrombocytopenia (HIT), caused by the development of antibodies against heparin/PF4 complexes. Here, we demonstrate the ability of heparin to induce neutrophil extracellular traps (NETs). NETs occurred with cell lysis and death, but live neutrophils releasing extracellular DNA strands, known as vital NETs, also occurred abundantly. Formation of NETs was time and dose dependent, and required reactive oxygen species and neutrophil elastase. Other compounds related to heparin such as low molecular weight heparin, fondaparinux and heparan sulfate either failed to induce NETs, or did so to a much lesser extent. Our findings suggest the ability of heparin to directly induce NET formation should be considered in the context of heparin treatment and HIT pathogenesis.
Subject(s)
Extracellular Traps/immunology , Extracellular Traps/metabolism , Heparin/metabolism , Leukocyte Elastase/metabolism , Thrombocytopenia/immunology , HumansABSTRACT
Influenza A virus (IAV) triggers the infected lung to produce IL-1 and recruit neutrophils. Unlike IL-1ß, however, little is known about IL-1α in terms of its mechanism of induction, action and physiological relevance to the host immunity against IAV infection. In particular, whether Z-DNA-binding protein 1 (ZBP1), a key molecule for IAV-induced cell death, is involved in the IL-1α induction, neutrophil infiltration and the physiological outcome has not been elucidated. Here, we show in a murine model that the IAV-induced IL-1α is mediated solely by ZBP1, in an NLRP3-inflammasome-independent manner, and is required for the optimal IL-1ß production followed by the formation of neutrophil extracellular traps (NETs). During IAV infection, ZBP1 displays a dual role in anti-IAV immune responses mediated by neutrophils, resulting in either protective or pathological outcomes in vivo. Thus, ZBP1-mediated IL-1α production is the key initial step of IAV-infected NETs, regulating the duality of the consequent lung inflammation.
Subject(s)
Inflammasomes/immunology , Inflammation/immunology , Influenza A virus/immunology , Interleukin-1alpha/immunology , Neutrophils/immunology , RNA-Binding Proteins/immunology , Animals , Dogs , Inflammation/metabolism , Inflammation/pathology , Interleukin-1alpha/metabolism , Lung Diseases/immunology , Lung Diseases/microbiology , Lung Diseases/pathology , Mice , Mice, Inbred C57BL , Mice, Knockout , Neutrophils/pathologyABSTRACT
Extracellular host-derived DNA, as one of damage associated molecular patterns (DAMPs), is associated with allergic type 2 immune responses. Immune recognition of such DNA generates the second messenger cyclic GMP-AMP (cGAMP) and induces type-2 immune responses; however, its role in allergic diseases, such as asthma, has not been fully elucidated. This study aimed to determine whether cGAMP could induce asthma when used as an adjuvant. We intranasally sensitized mice with cGAMP together with house dust mite antigen (HDM), followed by airway challenge with HDM. We then assessed the levels of eosinophils in the broncho-alveolar lavage fluid (BALF) and serum HDM-specific antibodies. cGAMP promoted HDM specific allergic asthma, characterized by significantly increased HDM specific IgG1 and total IgE in the serum and infiltration of eosinophils in the BALF. cGAMP stimulated lung fibroblast cells to produce IL-33 in vitro, and mice deficient for IL-33 or IL-33 receptor (ST2) failed to develop asthma enhancement by cGAMP. Not only Il-33-/- mice, but also Sting-/-, Tbk1-/-, and Irf3-/-Irf7-/- mice which lack the cGAMP-mediated innate immune activation failed to increase eosinophils in the BALF than that from wild type mice. Consistently, intranasal and oral administration of amlexanox, a TBK1 inhibitor, decreased cGAMP-induced lung allergic inflammation. Thus, cGAMP functions as a type 2 adjuvant in the lung and can promote allergic asthma in manners that dependent on the intracellular STING/TBK1/IRF3/7 signaling pathway and the resultant intercellular signaling pathway via IL-33 and ST2 might be a novel therapeutic target for allergic asthma.
Subject(s)
Aminopyridines/pharmacology , Asthma/drug therapy , Asthma/immunology , Interleukin-33/immunology , Nucleotides, Cyclic/immunology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/immunology , Allergens/drug effects , Allergens/immunology , Animals , Bronchoalveolar Lavage Fluid/immunology , Eosinophils/drug effects , Eosinophils/immunology , Female , Immunoglobulin E/immunology , Immunoglobulin G/immunology , Lung/drug effects , Lung/immunology , Mice , Mice, Inbred C57BL , Pulmonary Eosinophilia/drug therapy , Pulmonary Eosinophilia/immunology , Pyroglyphidae/immunology , Signal Transduction/drug effects , Signal Transduction/immunologyABSTRACT
Adjuvants improve the potency of vaccines, but the modes of action (MOAs) of most adjuvants are largely unknown. TLR-dependent and -independent innate immune signaling through the adaptor molecule MyD88 has been shown to be pivotal to the effects of most adjuvants; however, MyD88's involvement in the TLR-independent MOAs of adjuvants is poorly understood. Here, using the T-dependent antigen NIPOVA and a unique particulate adjuvant called synthetic hemozoin (sHZ), we show that MyD88 is required for early GC formation and enhanced antibody class-switch recombination (CSR) in mice. Using cell-type-specific MyD88 KO mice, we found that IgG2c class switching, but not IgG1 class switching, was controlled by B cell-intrinsic MyD88 signaling. Notably, IFN-γ produced by various cells including T cells, NK cells, and dendritic cells was the primary cytokine for IgG2c CSR and B-cell intrinsic MyD88 is required for IFN-γ production. Moreover, IFN-γ receptor (IFNγR) deficiency abolished sHZ-induced IgG2c production, while recombinant IFN-γ administration successfully rescued IgG2c CSR impairment in mice lacking B-cell intrinsic MyD88. Together, our results show that B cell-intrinsic MyD88 signaling is involved in the MOA of certain particulate adjuvants and this may enhance our specific understanding of how adjuvants and vaccines work.
Subject(s)
B-Lymphocytes/immunology , Immunoglobulin Class Switching/immunology , Immunoglobulin G/immunology , Interferon-gamma/immunology , Myeloid Differentiation Factor 88/immunology , Signal Transduction/immunology , Adjuvants, Immunologic/pharmacology , Animals , Dendritic Cells/immunology , Killer Cells, Natural/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , T-Lymphocytes/immunologyABSTRACT
Neutrophil extracellular trap (NET) formation involves the release of DNA outside the cell to neutralize pathogens. Techniques such as live microscopy, flow cytometry, and intravital imaging allow the characterization of NETs, but these either cannot be applied in vivo, lack specificity or require invasive procedures. We developed an automated analysis method to rapidly acquire and characterize cells as NETs or NET precursors, as opposed to cells undergoing other forms of cell death, using imaging flow cytometry. NETs were maintained in solution using a novel three-dimensional cell culture system in which cells are suspended at the interface of two liquids of different density. Critically, we identify NETs using an image analysis algorithm based on morphological data showing the extrusion of DNA beyond the cell boundaries. In vitro, we used this technique to demonstrate different requirements for NET formation in human and mouse neutrophils. We also measured NETs in whole blood during infection of mice with the malaria parasite Plasmodium yoelii. We expect this technique will provide a valuable approach to better understand the process of NET formation and its importance in disease. © 2019 International Society for Advancement of Cytometry.
Subject(s)
Extracellular Traps/metabolism , Image Cytometry/methods , Algorithms , Animals , Apoptosis/drug effects , Automation , Extracellular Traps/drug effects , Image Processing, Computer-Assisted , Kinetics , Lipopolysaccharides/pharmacology , Mice , Mice, Inbred C57BL , NADPH Oxidases/metabolism , Neutrophils/drug effects , Neutrophils/metabolism , Protein-Arginine Deiminases/metabolism , Tetradecanoylphorbol Acetate/pharmacology , Time FactorsABSTRACT
Recently, it was reported that 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CyD), a common pharmaceutical additive, can act as a vaccine adjuvant to enhance protective type-2 immunogenicity to co-administered seasonal influenza split vaccine by inducing host-derived damage-associated molecular patterns (DAMPs). However, like most other DAMP-inducing adjuvants such as aluminum hydroxide (Alum), HP-ß-CyD may not be sufficient for the induction of protective type-1 (cellular) immune responses, thereby leaving room for improvement. Here, we demonstrate that a combination of HP-ß-CyD with a humanized TLR9 agonist, K3 CpG-ODN, a potent pathogen-associated molecular pattern (PAMP), enhanced the protective efficacy of the co-administered influenza split vaccine by inducing antigen-specific type-2 and type-1 immune responses, respectively. Moreover, substantial antigen-specific IgE induction by HP-ß-CyD, which can cause an allergic response to immunized antigen was completely suppressed by the addition of K3 CpG-ODN. Furthermore, HP-ß-CyD- and K3 CpG-ODN-adjuvanted influenza split vaccination protected the mice against lethal challenge with high doses of heterologous influenza virus, which could not be protected against by single adjuvant vaccines. Further experiments using gene deficient mice revealed the unique immunological mechanism of action in vivo, where type-2 and type-1 immune responses enhanced by the combined adjuvants were dependent on TBK1 and TLR9, respectively, indicating their parallel signaling pathways. Finally, the analysis of immune responses in the draining lymph node suggested that HP-ß-CyD promotes the uptake of K3 CpG-ODN by plasmacytoid dendritic cells and B cells, which may contributes to the activation of these cells and enhanced production of IgG2c. Taken together, the results above may offer potential clinical applications for the combination of DAMP-inducing adjuvant and PAMP adjuvant to improve vaccine immunogenicity and efficacy by enhancing both type-2 and type-1 immune responses in a parallel manner.
Subject(s)
B-Lymphocytes/immunology , Dendritic Cells/immunology , Influenza A Virus, H1N1 Subtype/physiology , Influenza Vaccines/immunology , Influenza, Human/immunology , Orthomyxoviridae Infections/immunology , Th1 Cells/immunology , Th2 Cells/immunology , 2-Hydroxypropyl-beta-cyclodextrin/immunology , Adjuvants, Immunologic , Alarmins/metabolism , Animals , Antibodies, Viral/blood , Cells, Cultured , Female , Humans , Immunogenicity, Vaccine , Lymphocyte Activation , Mice , Mice, Inbred C57BL , Mice, Knockout , Oligodeoxyribonucleotides/immunology , Pathogen-Associated Molecular Pattern Molecules/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Toll-Like Receptor 9/agonists , Toll-Like Receptor 9/genetics , VaccinationABSTRACT
BACKGROUND: Malaria is known to cause acute and deadly complications. However, malaria can cause unforeseen pathologies due to its chronicity. It increases the risk of endemic Burkitt Lymphoma development by inducing DNA damage in germinal centre (GC) B cells, and leading higher frequency of Epstein-Barr virus (EBV)-infected cells in GCs. EBV is well known for its tropism for B cells. However, less is known about EBV's interaction with T cells and its association with T cell lymphoma. CASE PRESENTATION: A 43-year-old Sudanese male admitted to hospital in Istanbul, Turkey, a non-endemic country, with hyperpigmented painful skin rashes on his whole body. A complete blood count and a peripheral blood smear during admission revealed large granular lymphocytes (LGLs) with abnormally higher CD8 T cell numbers. Additional skin biopsy and pathology results were compatible with CD8+ T cell lymphoproliferative disorder with skin involvement. Patient was treated and discharged. However, a pathologist noticed unusual structures in skin tissue samples. Careful evaluation of skin biopsy samples by polarized microscopy revealed birefringent crystalloid structures resembling malarial haemozoin mainly loaded in macrophages and giant histiocytes. After purification of DNA from the skin biopsy samples, nested PCR was performed for the detection of Plasmodium parasites and Plasmodium falciparum DNA was amplified. Because, the co-presence of EBV infection with malaria is a well-known aetiology of lymphoma, EBV-early RNA (EBER) transcripts were investigated in paraffin-embedded tissue samples and found to be positive in macrophage-like histiocytes. CONCLUSIONS: This is a unique case of malaria and EBV infection in a T-LGL lymphoma patient who presented in a non-endemic country. This case emphasizes the clinical importance of EBV monitoring in T-LGL patients with skin involvement. Notably, Plasmodium infection should be examined in patients from malaria endemic regions by pathological and molecular investigations.