Bovine β-lactoglobulin-induced passive systemic anaphylaxis model using humanized NOG hIL-3/hGM-CSF transgenic mice

Food allergy is a common disease caused by intake of allergen-containing foods, such as milk, eggs, peanuts and wheat. Systemic anaphylaxis is a severe hypersensitive allergic reaction resulting from degranulation of mast cells or basophils after cross-linking of surface high-affinity IgE receptors (Fcε-RI) with allergen-specific IgE and allergens. In this study, we developed a novel human mast cell/basophil-engrafted mouse model that recapitulates systemic anaphylaxis triggered by β-lactoglobulin (BLG), a major allergen found in cow’s milk. Human CD34+ hematopoietic stem cells were transferred into NOG (non-Tg) or NOG hIL-3/hGM-CSF transgenic (Tg) mice. After 14–16 weeks, bovine BLG-specific human IgE was intravenously injected into humanized mice, followed by intravenous or oral bovine BLG exposure 1 day later. Body temperature in Tg, but not in non-Tg, mice gradually decreased within 10 min, and 80% of Tg mice died within 1 h by intravenous BLG exposure. Serum histamine levels and anaphylaxis scores in Tg mice were markedly increased compared to non-Tg mice. Furthermore, these allergic symptoms were significantly inhibited by epinephrine treatment of the Tg mice. Therefore, the current NOG hIL-3/hGM-CSF Tg mouse model may be useful for development of novel anaphylaxis drugs for treatment of food allergies and for safety assessment of low-allergenicity extensively hydrolyzed cow’s milk whey protein-based infant formulas.

TPC1 deficiency or blockade augments systemic anaphylaxis and mast cell activity

Mast cells and basophils are main drivers of allergic reactions and anaphylaxis, for which prevalence is rapidly increasing. Activation of these cells leads to a tightly controlled release of inflammatory mediators stored in secretory granules. The release of these granules is dependent on intracellular calcium (Ca2+) signals. Ca2+release from endolysosomal compartments is mediated via intracellular cation channels, such as two-pore channel (TPC) proteins. Here, we uncover a mechanism for how TPC1 regulates Ca2+homeostasis and exocytosis in mast cells in vivo and ex vivo. Notably, in vivo TPC1 deficiency in mice leads to enhanced passive systemic anaphylaxis, reflected by increased drop in body temperature, most likely due to accelerated histamine-induced vasodilation. Ex vivo, mast cell-mediated histamine release and degranulation was augmented upon TPC1 inhibition, although mast cell numbers and size were diminished. Our results indicate an essential role of TPC1 in endolysosomal Ca2+uptake and filling of endoplasmic reticulum Ca2+stores, thereby regulating exocytosis in mast cells. Thus, pharmacological modulation of TPC1 might blaze a trail to develop new drugs against mast cell-related diseases, including allergic hypersensitivity.

Increased Urinary 3-Mercaptolactate Excretion and Enhanced Passive Systemic Anaphylaxis in Mice Lacking Mercaptopyruvate Sulfurtransferase, a Model of Mercaptolactate-Cysteine Disulfiduria

Mercaptopyruvate sulfurtransferase (Mpst) and its homolog thiosulfate sulfurtransferase (Tst = rhodanese) detoxify cyanide to thiocyanate. Mpst is attracting attention as one of the four endogenous hydrogen sulfide (H2S)/reactive sulfur species (RSS)-producing enzymes, along with cystathionine β-synthase (Cbs), cystathionine γ-lyase (Cth), and cysteinyl-tRNA synthetase 2 (Cars2). MPST deficiency was found in 1960s among rare hereditary mercaptolactate-cysteine disulfiduria patients. Mpst-knockout (KO) mice with enhanced liver Tst expression were recently generated as its model; however, the physiological roles/significances of Mpst remain largely unknown. Here we generated three independent germ lines of Mpst-KO mice by CRISPR/Cas9 technology, all of which maintained normal hepatic Tst expression/activity. Mpst/Cth-double knockout (DKO) mice were generated via crossbreeding with our previously generated Cth-KO mice. Mpst-KO mice were born at the expected frequency and developed normally like Cth-KO mice, but displayed increased urinary 3-mercaptolactate excretion and enhanced passive systemic anaphylactic responses when compared to wild-type or Cth-KO mice. Mpst/Cth-DKO mice were also born at the expected frequency and developed normally, but excreted slightly more 3-mercaptolactate in urine compared to Mpst-KO or Cth-KO mice. Our Mpst-KO, Cth-KO, and Mpst/Cth-DKO mice, unlike semi-lethal Cbs-KO mice and lethal Cars2-KO mice, are useful tools for analyzing the unknown physiological roles of endogenous H2S/RSS production.

Anaphylaxis stimulates afferent vagal nerve activity and efferent sympathetic nerve activity in the stomach of anesthetized rats

Systemic anaphylaxis is a life-threatening and allergic reaction that affects various organs. We previously reported that, in the stomach, gastric vasoconstriction occurring at the late phase (15–55 min after injection of ovalbumin antigen) was observed in anesthetized rats sensitized with ovalbumin. In addition, anaphylaxis enhances gastric motility and delays emptying. However, the role of extrinsic autonomic nervous system on antigen-induced gastric alterations was not known. Thus, using the same rat anaphylaxis model, we aimed to determine the changes in the efferent and afferent autonomic nerve activities in the stomach during anaphylactic hypotension. The findings showed that injection of ovalbumin antigen caused substantial systemic hypotension in all sensitized rats. The efferent gastric sympathetic nerve activity (ef-GSNA), but not the efferent vagal nerve activity, increased only at the early phase (1–10 min after injection of ovalbumin antigen) and showed baroreceptor reflex, as evidenced by a stimulatory response to sodium nitroprusside-induced hypotension. In general, excitation of ef-GSNA could induce pylorus sphincter contraction and gastric vasoconstriction. In the present study, we found that sympathectomy attenuated the anaphylaxis-induced decrease in gastric flux but not the increase in gastric vascular resistance. Thus, the increase in ef-GSNA may cause anaphylactic pylorus sphincter contraction but not anaphylactic gastric vasoconstriction. On the other hand, the afferent gastric vagal nerve activity, but not the afferent sympathetic nerve activity, increased during the early phase of anaphylactic hypotension. However, vagotomy produced no effects on the anaphylactic gastric dysfunction. In conclusion, the gastric sympathetic nerves partly modulate stomach function during systemic anaphylaxis.