Cromolyn, widely characterized as a ‘mast cell stabilizer', has been used in mice to investigate the biological roles of mast cells in vivo. However, it is not clear to what extent cromolyn can ...either limit the function of mouse mast cells or influence biological processes in mice independently of effects on mast cells. We confirmed that cromolyn (at 10 mg/kg in vivo or 10–100 μM in vitro) can inhibit IgE-dependent mast cell activation in rats in vivo (measuring Evans blue extravasation in passive cutaneous anaphylaxis (PCA) and increases in plasma histamine in passive systemic anaphylaxis (PSA)) and in vitro (measuring peritoneal mast cell (PMC) β-hexosaminidase release and prostaglandin D2 synthesis). However, under the conditions tested, cromolyn did not inhibit those mast cell-dependent responses in mice. In mice, cromolyn also failed to inhibit the ear swelling or leukocyte infiltration at sites of PCA. Nor did cromolyn inhibit IgE-independent degranulation of mouse PMCs induced by various stimulators in vitro. At 100 mg/kg, a concentration 10 times higher than that which inhibited PSA in rats, cromolyn significantly inhibited the increases in plasma concentrations of mouse mast cell protease-1 (but not of histamine) during PSA, but had no effect on the reduction in body temperature in this setting. Moreover, this concentration of cromolyn (100 mg/kg) also inhibited LPS-induced TNF production in genetically mast cell-deficient C57BL/6-KitW-sh/W-sh mice in vivo. These results question cromolyn's effectiveness and selectivity as an inhibitor of mast cell activation and mediator release in the mouse.
Understanding tissue structure and function requires tools that quantify the expression of multiple proteins while preserving spatial information. Here, we describe MIBI-TOF (multiplexed ion beam ...imaging by time of flight), an instrument that uses bright ion sources and orthogonal time-of-flight mass spectrometry to image metal-tagged antibodies at subcellular resolution in clinical tissue sections. We demonstrate quantitative, full periodic table coverage across a five-log dynamic range, imaging 36 labeled antibodies simultaneously with histochemical stains and endogenous elements. We image fields of view up to 800 μm × 800 μm at resolutions down to 260 nm with sensitivities approaching single-molecule detection. We leverage these properties to interrogate intrapatient heterogeneity in tumor organization in triple-negative breast cancer, revealing regional variability in tumor cell phenotypes in contrast to a structured immune response. Given its versatility and sample back-compatibility, MIBI-TOF is positioned to leverage existing annotated, archival tissue cohorts to explore emerging questions in cancer, immunology, and neurobiology.
Thirdhand smoke (THS) represents the accumulation of secondhand smoke on indoor surfaces and in dust, which, over time, can become more toxic than secondhand smoke. Although it is well known that ...children of smokers are at increased risk for asthma or asthma exacerbation if the disease is already present, how exposure to THS can influence the development or exacerbation of asthma remains unknown.
We investigated whether epicutaneous exposure to an important component of THS, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), can influence asthma pathology in a mouse model elicited by means of repeated intranasal challenge with cockroach antigen (CRA).
Wild-type mice, α7 nicotinic acetylcholine receptor (nAChR)– or mast cell (MC)–deficient mice, and mice with MCs that lacked α7 nAChRs or were the host's sole source of α7 nAChRs were subjected to epicutaneous NNK exposure, intranasal CRA challenge, or both, and the severity of features of asthma pathology, including airway hyperreactivity, airway inflammation, and airway remodeling, was assessed.
We found that α7 nAChRs were required to observe adverse effects of epicutaneous NNK exposure on multiple features of CRA-induced asthma pathology. Moreover, MC expression of α7 nAChRs contributed significantly to the ability of epicutaneous NNK exposure to exacerbate airway hyperreactivity to methacholine, airway inflammation, and airway remodeling in this model.
Our results show that skin exposure to NNK, a component of THS, can exacerbate multiple features of a CRA-induced model of asthma in mice and define MCs as key contributors to these adverse effects of NNK.
There is evidence that mast cells, basophils, and IgE can contribute to immune responses to parasites; however, the relative levels of importance of these effector elements in parasite immunity are ...not fully understood. Previous work in
-deficient and c-
mutant
mice indicated that interleukin-3 and c-Kit contribute to expulsion of the intestinal nematode
during primary infection. Our findings in mast cell-deficient
mice and two types of mast cell-deficient mice that have normal c-
("Hello
ty" and MasTRECK mice) confirmed prior work in
mice that suggested that mast cells play an important role in
egg clearance in primary infections. We also assessed a possible contribution of basophils in immune responses to
By immunohistochemistry, we found that numbers of basophils and mast cells were markedly increased in the jejunal mucosa during primary infections with
Studies in basophil-deficient
mice revealed a small but significant contribution of basophils to
egg clearance in primary infections. Studies in mice deficient in various components of immune responses showed that CD4
T cells and ILC2 cells, IgG, FcRγ, and, to a lesser extent, IgE and FcεRI contribute to effective immunity in primary
infections. These findings support the conclusion that the hierarchy of importance of immune effector mechanisms in primary
infection is as follows: CD4
T cells/ILC2 cells, IgG, and FcRγ>mast cells>IgE and FcεRI>basophils. In contrast, in secondary
infection, our evidence indicates that the presence of CD4
T cells is of critical importance but mast cells, antibodies, and basophils have few or no nonredundant roles.
Allergen immunotherapy can desensitize even subjects with potentially lethal allergies, but the changes induced in T cells that underpin successful immunotherapy remain poorly understood. In a cohort ...of peanut-allergic participants, we used allergen-specific T-cell sorting and single-cell gene expression to trace the transcriptional “road-map” of individual CD4+ T cells throughout immunotherapy. We found that successful immunotherapy induces allergen-specific CD4+ T cells to expand and shift toward an “anergic” Th2 T-cell phenotype largely absent in both pretreatment participants and healthy controls. These findings show that sustained success, even after immunotherapy is withdrawn, is associated with the induction, expansion, and maintenance of immunotherapy-specific memory and naive T-cell phenotypes as early as 3 mo into immunotherapy. These results suggest an approach for immune monitoring participants undergoing immunotherapy to predict the success of future treatment and could have implications for immunotherapy targets in other diseases like cancer, autoimmune disease, and transplantation.
Background Mast cells have gained notoriety based on their detrimental contributions to IgE-mediated allergic disorders. Although mast cells express the vitamin D receptor (VDR), it is not clear to ...what extent 1α,25-dihydroxyvitamin D3 (1α,25OH2 D3 ) or its predominant inactive precursor metabolite in the circulation, 25-hydroxyvitamin D3 (25OHD3 ), can influence IgE-mediated mast cell activation and passive cutaneous anaphylaxis (PCA) in vivo. Objective We sought to assess whether the vitamin D3 metabolites 25OHD3 and 1α,25(OH)2 D3 can repress IgE-dependent mast cell activation through mast cell–25-hydroxyvitamin D-1α-hydroxylase (CYP27B1) and mast cell–VDR activity. Methods We measured the extent of vitamin D3 suppression of IgE-mediated mast cell degranulation and mediator production in vitro , as well as the vitamin D3 –induced curtailment of PCA responses in WBB6F1 - KitW/W-v or C57BL/6J- KitW-sh/W-sh mice engrafted with mast cells that did or did not express VDR or CYP27B1. Results Here we show that mouse and human mast cells can convert 25OHD3 to 1α,25(OH)2 D3 through CYP27B1 activity and that both of these vitamin D3 metabolites suppressed IgE-induced mast cell–derived proinflammatory and vasodilatory mediator production in a VDR-dependent manner in vitro . Furthermore, epicutaneously applied vitamin D3 metabolites significantly reduced the magnitude of skin swelling associated with IgE-mediated PCA reactions in vivo ; a response that required functional mast cell–VDRs and mast cell–CYP27B1. Conclusion Taken together, our findings provide a mechanistic explanation for the anti-inflammatory effects of vitamin D3 on mast cell function by demonstrating that mast cells can actively metabolize 25OHD3 to dampen IgE-mediated mast cell activation in vitro and in vivo.
Background Studies with c- kit mutant mast cell (MC)–deficient mice and antibody-mediated depletion of basophils suggest that both MCs and basophils can contribute to peanut-induced anaphylaxis ...(PIA). However, interpretation of data obtained by using such approaches is complicated because c- kit mutant mice have several phenotypic abnormalities in addition to MC deficiency and because basophil-depleting antibodies can also react with MCs. Objective We analyzed (1) the changes in the features of PIA in mice after the selective and inducible ablation of MCs or basophils and (2) the possible importance of effector cells other than MCs and basophils in the PIA response. Methods Wild-type and various mutant mice were orally sensitized with peanut extract and cholera toxin weekly for 4 weeks and challenged intraperitoneally with peanut extract 2 weeks later. Results Peanut-challenged, MC-deficient Kit W-sh/W-sh mice had reduced immediate hypothermia, as well as a late-phase decrease in body temperature that was abrogated by antibody-mediated depletion of neutrophils. Diphtheria toxin–mediated selective depletion of MCs or basophils in Mcpt5-Cre ; iDTR and Mcpt8 DTR mice, respectively, and treatment of wild-type mice with the basophil-depleting antibody Ba103 significantly reduced peanut-induced hypothermia. Non–c- kit mutant MC- and basophil-deficient Cpa3-Cre;Mcl-1 fl/fl mice had reduced but still significant responses to peanut. Conclusion Inducible and selective ablation of MCs or basophils in non–c- kit mutant mice can significantly reduce PIA, but partial responses to peanut can still be observed in the virtual absence of both cell types. The neutrophilia in Kit W-sh/W-sh mice might influence the responses of these mice in this PIA model.
Basophils are evolutionarily conserved in vertebrates, despite their small numbers and short life span, suggesting that they have beneficial roles in maintaining health. However, these roles are not ...fully defined. Here we demonstrate that basophil-deficient mice exhibit reduced bacterial clearance and increased morbidity and mortality in the cecal ligation and puncture (CLP) model of sepsis. Among the several proinflammatory mediators that we measured, tumor necrosis factor (TNF) was the only cytokine that was significantly reduced in basophil-deficient mice after CLP. In accordance with that observation, we found that mice with genetic ablation of Tnf in basophils exhibited reduced systemic concentrations of TNF during endotoxemia. Moreover, after CLP, mice whose basophils could not produce TNF, exhibited reduced neutrophil and macrophage TNF production and effector functions, reduced bacterial clearance, and increased mortality. Taken together, our results show that basophils can enhance the innate immune response to bacterial infection and help prevent sepsis.
Most secreted growth factors and cytokines are functionally pleiotropic because their receptors are expressed on diverse cell types. While important for normal mammalian physiology, pleiotropy limits ...the efficacy of cytokines and growth factors as therapeutics. Stem cell factor (SCF) is a growth factor that acts through the c-Kit receptor tyrosine kinase to elicit hematopoietic progenitor expansion but can be toxic when administered in vivo because it concurrently activates mast cells. We engineered a mechanism-based SCF partial agonist that impaired c-Kit dimerization, truncating downstream signaling amplitude. This SCF variant elicited biased activation of hematopoietic progenitors over mast cells in vitro and in vivo. Mouse models of SCF-mediated anaphylaxis, radioprotection, and hematopoietic expansion revealed that this SCF partial agonist retained therapeutic efficacy while exhibiting virtually no anaphylactic off-target effects. The approach of biasing cell activation by tuning signaling thresholds and outputs has applications to many dimeric receptor-ligand systems.
•An engineered variant of stem cell factor acts as partial agonist of c-Kit•SCF partial agonist exhibits biased activation of HSPCs versus mast cells•Partial agonist of c-Kit retains therapeutic efficacy but mitigates toxicity in vivo
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A ligand engineering strategy is used to amplify hematopoietic stem cells but avoid unwanted off-target effects mediated by mast cells.
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