Levels of 15-HETE and TXB2 in exhaled breath condensates as markers for diagnosis of childhood asthma and its therapeutic outcome.

BACKGROUND: Dysregulation of eicosanoids is associated with asthma and a composite of oxylipins, including exhaled leukotriene B4 (LTB4 ), characterizes childhood asthma. While fractional exhaled nitric oxide (FeNO) has been used as the standard for monitoring steroid responsiveness, the potential utility of eicosanoids in monitoring the therapeutic outcomes remains unclear. We aimed to examine the levels of major eicosanoids representing different metabolic pathways in exhaled breath condensates (EBCs) of children with asthma during exacerbation and after treatment. METHODS: Levels of 6 exhaled eicosanoid species in asthmatic children and healthy subjects were evaluated using ELISA. RESULTS: In addition to those previously reported, including LTB4 , the levels of exhaled 15-hydroxyeicosatetraenoic acid (15-HETE), but not thromboxane B2 (TXB2 ), showed significant difference between asthmatics (N = 318) and healthy controls (N = 97), particularly the severe group showed the lowest levels of exhaled 15-HETE. Receiver operating characteristic (ROC) curve analyses revealed similar distinguishing power for the levels of 15-HETE, FEV1 (forced expiratory volume in the first second), and FeNO, while the 15-HETE/LTB4 ratio was significantly lower in subjects with asthma as compared to that of healthy controls (p < 0.0001). Analysis of asthmatics (N = 75) during exacerbation and convalescence showed significant improvement in lung function (FEV1 , p < .001), but not FeNO, concomitant with significantly increased levels of 15-HETE (p < .001) and reduced levels of TXB2 (p < .05) at convalescence, particularly for those who at the top 30% level during exacerbation. Further, decreased LTB4 and lipoxin A4 (LXA4 ) at convalescence were noted only in those at the top 30 percentile during exacerbation. CONCLUSION: The exhaled 15-HETE was found to discriminate childhood asthma while decreased levels of exhaled TXB2 and increased levels of 15-HETE were prominent at convalescence.

Aryl hydrocarbon receptor signaling promotes ORMDL3-dependent generation of sphingosine-1-phosphate by inhibiting sphingosine-1-phosphate lyase.

Aryl hydrocarbon receptor (AhR), a cellular chemical sensor, controls cellular homeostasis, and sphingosine-1-phosphate (S1P), a bioactive intermediate of sphingolipid metabolism, is believed to have a role in immunity and inflammation, but their potential crosstalk is currently unknown. We aimed to determine whether there is a functional linkage between AhR signaling and sphingolipid metabolism. We showed that AhR ligands, including an environmental polycyclic aromatic hydrocarbon (PAH), induced S1P generation, and inhibited S1P lyase (S1PL) activity in resting cells, antigen/IgE-activated mast cells, and mouse lungs exposed to the AhR ligand alone or in combination with antigen challenge. The reduction of S1PL activity was due to AhR-mediated oxidation of S1PL at residue 317, which was reversible by the addition of an antioxidant or in cells with knockdown of the ORMDL3 gene encoding an ER transmembrane protein, whereas C317A S1PL mutant-transfected cells were resistant to the AhR-mediated effect. Furthermore, analysis of AhR ligand-treated cells showed a time-dependent increase of the ORMDL3-S1PL complex, which was confirmed by FRET analysis. This change increased the S1P levels, which in turn, induced mast cell degranulation via S1PR2 signaling. In addition, elevated levels of plasma S1P were found in children with asthma compared to non-asthmatic subjects. These results suggest a new regulatory pathway whereby the AhR-ligand axis induces ORMDL3-dependent S1P generation by inhibiting S1PL, which may contribute to the expression of allergic diseases.

Correction to: Aryl hydrocarbon receptor signaling promotes ORMDL3-dependent generation of sphingosine-1-phosphate by inhibiting sphingosine-1-phosphate lyase.

In this article, published online 23 March 2018, the affiliation 10 of Zhou Y was incorrect. The affiliation should be "Children's Hospital and Institute of Biomedical Sciences, Fudan University. Key Laboratory of Neonatal Disease, Ministry of Health, 201102, Shanghai, China." The authors regret the errors.

Fetal Phagocytes Take up Allergens to Initiate T-Helper Cell Type 2 Immunity and Facilitate Allergic Airway Responses.

RATIONALE: Actively acquired tolerance occurs when foreign antigens come into contact with the immature fetal immune system. OBJECTIVES: Armed with the knowledge of actively acquired tolerance, we attempted to prenatally abolish or diminish allergic responses. METHODS: In utero injection of adjuvant-free ovalbumin (OVA) was conducted in Gestational Day 14 FVB/N mouse fetuses. Postnatally, mice were evaluated for their resistance to intraperitoneal OVA sensitization and oral or aerosolized OVA challenge, and then they were examined for humoral and cellular immunological profiles, airway hyperresponsiveness to bronchospastic stimuli, and lung histology. Fluorescent conjugates of OVA were used for further studies of mechanisms. MEASUREMENTS AND MAIN RESULTS: This presumed tolerogenic action turned out to be a sensitization process with the development of anaphylaxis or heightened recall, T-helper cell type 2-skewed responses to postnatal encounter with OVA. Further postnatal aerosolized OVA stress triggered allergic lungs with functional and structural alterations of airways. The unintended consequence resulted from macrophage-like fetal phagocytes that took up OVA and differentiated toward dendritic cells. These fetal dendritic cell progenitors attenuated proteolysis of endocytosed OVA for delayed presentation in postnatal life. This specialty of fetal phagocytes effectively retains the memory of antigens internalized early before full development of the immune system, leading to an event of in utero sensitization. CONCLUSIONS: Our results have mechanical implications for prenatal imprinting of atopy and shed light on the importance of fetal phagocytes in shaping the developing immune system and initiating allergic airway diseases.

Postnatal donor lymphocytes enhance prenatally-created chimerism at the risk of graft-versus-host disease.

The major barrier to clinical application of in utero hematopoietic stem cell transplantation is insufficient chimerism for phenotypic correction of target diseases or induction of graft tolerance. Postnatal donor lymphocyte infusion (DLI) may enhance donor cell levels so as to further facilitate tolerance induction. We created murine mixed chimeras in utero. Chimeras with <10% donor cells were subjected to postnatal DLI to evaluate the effects of DLI on chimerism augmentation and skin tolerance induction. Within one day after DLI, recipients experienced a transient peaking of donor chimerism, which could be as high as 20~40%. However, the transient chimerism peaking didn't benefit donor skin survivals despite immediate skin placement after DLI. In case of fruitful DLI, chimerism augmentation was usually observed after a latent period of 2~4 weeks. Otherwise, chimerism would return to around pre-DLI levels by days 7~14. Peripheral chimerism of >3% could be consistently boosted up to >10%, whereas chimerism of <0.2% hardly showed any significant enhancement. As for chimerism levels of 0.2~3%, chimerism augmentation up to >10% succeeded in 3(15%) of 20 recipients. Notably, chimerism augmentation by postnatal DLI was often associated with unexpected death or graft-versus-host disease (GVHD). In conclusion, transient chimerism augmentation by DLI played no role in facilitating graft tolerance. Substantial augmentation by DLI demanded a threshold chimerism level and posed a serious risk of GVHD to the recipients. It raised the concern about using postnatal DLI to broaden therapeutic horizons of in utero hematopoietic stem cell transplantation.

Recipients with in utero induction of tolerance upregulated MHC class I in the engrafted donor skin.

The alterations in MHC class I expression play a crucial step in immune evasion of cancer or virus-infected cells. This study aimed to examine whether tolerized grafts modified MHC class I expression. FVB/N mice were rendered tolerant of C57BL/6 alloantigens by in utero transplantation of C57BL/6 marrows. Postnatally, engrafted donor skins and leukocytes were examined for their MHC expression by quantitative real-time PCR and flow cytometry. Engrafted donor skins upregulated their MHC class I related gene transcripts after short-term (1~2 weeks) or long-term (>1 month) engraftment. This biological phenomenon was simultaneously associated with upregulation of TAP1 gene transcripts, suggesting an important role of TAP1 in the regulation of MHC class I pathway. The surface MHC class I molecules of H-2K(b) in engrafted donor leukocytes consistently showed overexpression. Conclusively, the induction of allograft tolerance involved biological modifications of donor transplants. The overexpression of MHC class I within engrafted transplants of tolerant mice might be used as the tolerance biomarkers for identifying a state of graft tolerance.

Allogeneic lymphocytes exerted graft-versus-host rather than tolerogenic effects on preimmune fetuses.

BACKGROUND: Among cell suspensions from different origins, lymphocytes were reported to have the superiority of tolerance-conferring capacity in preimmune hosts. However, this belief was derived directly from murine combinations with fewer major histocompatibility complex (MHC) barriers that are exceptional in the clinical arena. Because of the potential for prenatal tolerance induction to facilitate postnatal therapies, it is important to examine the relative merits and hazards of fully MHC-mismatched naïve lymphocytes as the prenatal tolerogenic agent in the preimmune fetus to cross MHC barriers. MATERIALS AND METHODS: In utero injection of C57BL/6 splenic lymphocytes was conducted in gestational day 14 FVB/N fetuses. Then, FVB/N recipients were subjected to the evaluation of hematopoietic chimerism, donor-specific tolerance, and graft-versus-host disease (GVHD). RESULTS: With a dose of ≥ 5 × 10(5) C57BL/6 lymphocytes, the recipients born alive either died unexpectedly by maternal cannibalization or succumbed to GVHD within postnatal 1 mo. GVHD mice showed significant hematopoietic chimerism that was dominated by donor CD3 T cells. It was found that allogeneic lymphocytes could rapidly damage the fetal liver within 5 d after injection. Fetal recipients could survive a dose of ≤ 2 × 10(5) allogeneic lymphocytes beyond 1 mo of age, but at best showed microchimerism that was insufficient to confer donor-specific skin tolerance. CONCLUSIONS: Fully MHC-mismatched lymphocytes injected in utero had lethal graft-versus-host effects, which might rapidly develop within 1 wk after injection in preimmune fetuses. They were incapable of conferring significant hematopoietic chimerism and graft tolerance even at bearable doses.

Characterization of tolerance induction through prenatal marrow transplantation: the requirement for a threshold level of chimerism to establish rather than maintain postnatal skin tolerance.

Hematopoietic chimerism resulting from prenatal marrow transplantation does not consistently result in allotolerance for unidentified causes. In a C57BL/6-into-FVB/N murine model, we transplanted T-cell-depleted adult marrow on gestational day 14 to elucidate the immunological significance of chimerism towards postnatal tolerance. Postnatally, chimerism was examined by flow cytometry, and tolerance by skin transplantation and mixed lymphocyte reaction. Regulatory T cells were quantified by FoxP3 expression. Peripheral chimerism linearly related to thymic chimerism, and predicted the degree of graft acceptance with levels >3% at skin placement, yielding consistent skin tolerance. Low- and high-level chimeras had lower intrathymic CD3(high) expression than microchimeras or untransplanted mice. Regardless of the skin tolerance status in mixed chimeras, donor-specific alloreactivity by lymphocytes was suppressed but could be partially restored by exogenous interleukin-2. Recipients that lost peripheral chimerism did not accept donor skin unless prior donor skin had engrafted at sufficient chimerism levels, suggesting that complete tolerance can develop as a consequence of chimerism-related immunosuppression of host lymphocytes and the tolerogenic effects of donor skin. Thus, hematopoietic chimerism exerted immunomodulatory effects on the induction phase of allograft tolerance. Once established, skin tolerance did not fade away along with spontaneous regression of peripheral and tissue chimerism, as well as removal of engrafted donor skin. Neither did it break following in vivo depletion of increased regulatory T cells, and subcutaneous interleukin-2 injection beneath the engrafted donor skin. Those observations indicate that the maintenance of skin tolerance is multifaceted, neither solely dependent upon hematopoietic chimerism and engrafted donor skin nor on the effects of regulatory T cells or clonal anergy. We conclude that hematopoietic chimerism generated by in utero hematopoietic stem cell transplantation is critical to establish rather than maintain postnatal skin tolerance. Therefore, the diminution of hematopoietic chimerism below a threshold level does not nullify an existing tolerance state, but lessens the chance of enabling complete tolerance.