Christophe Frossard

APRIL secreted by neutrophils binds to heparan sulfate proteoglycans to create plasma cell niches in human mucosa

The bone marrow constitutes a favorable environment for long-lived antibody-secreting plasma cells, providing blood-circulating antibody. Plasma cells are also present in mucosa-associated lymphoid tissue (MALT) to mediate local frontline immunity, but how plasma cell survival there is regulated is not known. Here we report that a proliferation-inducing ligand (APRIL) promoted survival of human upper and lower MALT plasma cells by upregulating expression of the antiapoptotic proteins bcl-2, bcl-xL, and mcl-1. The in situ localization of APRIL was consistent with such a prosurvival role in MALT. In upper MALT, tonsillar epithelium produced APRIL. Upon infection, APRIL production increased considerably when APRIL-secreting neutrophils recruited from the blood infiltrated the crypt epithelium. Heparan sulfate proteoglycans (HSPGs) retained secreted APRIL in the subepithelium of the infected zone to create APRIL-rich niches, wherein IgG-producing plasma cells accumulated. In lower MALT, neutrophils were the unique source of APRIL, giving rise to similar niches for IgA-producing plasmocytes in villi of lamina propria. Furthermore, we found that mucosal humoral immunity in APRIL-deficient mice is less persistent than in WT mice. Hence, production of APRIL by inflammation-recruited neutrophils may create plasma cell niches in MALT to sustain a local antibody production.

Oral carrageenan induces antigen-dependent oral tolerance: prevention of anaphylaxis and induction of lymphocyte anergy in a murine model of food allergy.

Immunosuppressive effects of carrageenan, a high-molecular-weight polysaccharide, on antibody and T cell responses have been previously demonstrated. However, its effect on anaphylaxis is unknown. Our objectives were to test carrageenan-mediated oral tolerance induction in young mice subsequently sensitized to a common cows milk antigen. C3H/HeJ mice were fed or not λ-carrageenan (0.5 g/L) and/or 0.01 mg/mL β-lactoglobulin (BLG) for 5 d before oral sensitization with BLG and cholera toxin. Subsequently, the mice were challenged with BLG and symptom scores of anaphylaxis were recorded. Mesenteric lymph node cells, spleen cells, Peyers patches cells, intraepithelial lymphocytes, and lamina propria lymphocytes were isolated and stimulated in vitro with BLG, IL-2, or left unstimulated. BLG-specific IgG, IgG1, and IgG2a antibodies were measured. Pretreatment with carrageenan and BLG, but not pretreatment with either carrageenan or BLG alone or omission of pretreatment, diminished significantly the number of anaphylactic mice after BLG challenge (6.3%versus 53% in mice without pretreatment, p = 0.006). Mesenteric lymph nodes and spleen cells from pretreated mice proliferated less in presence of BLG or IL-2 than cells from sensitized control mice. Antigen-specific antibody production and passive cutaneous anaphylaxis was not suppressed by carrageenan and BLG pretreatment. In conclusion, carrageenan administered to young mice in conjunction with low doses of allergen before sensitization efficiently prevents anaphylaxis.

Skin tests and in vitro allergy tests have a poor diagnostic value for benign skin rashes due to β-lactams in children

To the Editor, Skin rashes are frequent in children treated by b-lactams (BL), with an estimated incidence ranging between 1% and 5% (1). In clinical practice, most of these children are labeled as ‘penicillin allergic’ without appropriate testing, mostly due to fear of lifethreatening reactions. However, an allergic reaction can be demonstrated by an oral provocation test (OPT) in <10% of the patients developing a rash while treated by BL (2–4). The cause is more likely to be related to the underlying infection rather than an allergic reaction to the antibiotic (3). Overdiagnosis of BL allergy constitutes a major public health problem by increasing health costs and by contributing to overall antibiotic resistance. Thus, an accurate diagnosis is considered as a major contribution to the cost-effective health cares and will be based on complete allergic workup (5). Delayed-reading intradermal tests (IDT) and lymphocyte transformation tests (LTT) have been proposed for a long time in the diagnosis of nonimmediate reactions to BL. Although several recent studies have highlighted the importance of the OPT, there are only few pediatric studies evaluating the added diagnostic value of currently available tests, that is, IDT and LTT. Based on the gold standard, that is, the OPT, the aim of the current study was to evaluate the diagnostic value of those tests in prospectively recruited children. We analyzed the data from 14 patients with a nonimmediate BL allergy confirmed by a positive OPT. Six patients were prospectively recruited at the emergency department of the Geneva University Hospital between 2006 and 2008 from an original study on BL allergy in children (3). Among 250 children challenged at our allergy outpatient’s clinic, eight additional patients with a positive OPT to BL were recruited prospectively. Eighty-two patients with a negative OPT from our previous prospective study were used as negative controls (3). The inclusion and exclusion criteria are described in the Appendix S1. The study was approved by local ethics committee, and informed consent was obtained before enrollment. All the included patients underwent a complete allergic workup including IDT to penicilloyl-polylysine (PPL), minor determinant mixture (MDM) (Diater, Spain), amoxicillin (AX) (Clamoxyl; GlaxoSmithKline, Switzerland) at 25 mg/ml, or cefuroxim (Zinacef ; GlaxoSmithKline, Switzerland) at 3 mg/ ml. Reading was performed both at 20 min and 48 h (3, 5). Regardless of the results of IDT, an OPT with the incriminated BL was performed in all patients as previously reported (3).Briefly, after receiving 150% of the standard dose, the patients were observed for 2 h in the hospital to exclude an immediate reaction. Then, the involved drug, at the therapeutic dose, was given at home for a further 48 h to all the patients without immediate reactions. The parents were contacted by phone at the end of the test. LTT were performed as previously described (6), by measuring (3) H-thymidine incorporation after 7 days of incubation with medium only, the incriminated antibiotic (i.e. amoxicillin or cefuroxim) added to the medium at concentrations ranging from 0.01 to 1 mg/ml, and phytohemagglutinin A (PHA) as a control mitogen. Stimulation Index (SI) >2.5 was regarded as positive (6). In addition, basophils activation tests (BAT) were performed according to the manufacturer’s instructions (Flowcast, Buhlmann, Switzerland), by measuring the proportion of activated basophils (CCR3CD63) (7). Minimum of 500 basophils were analyzed. To be considered positive, the SI (% of BL stimulation divided by % of negative control) must be >2 and the cutoff values for BL >7%. Characteristics of patients are shown in supplemental Table A1. The complete allergic workup occurred at a median of 2.4 months (25–75% interquartile range: 2–3.1 months) after the index event. Immediate-reading IDT were positive in seven patients (8.5%) with a negative OPT and in seven patients (50%) with a positive OPT. The culprit antibiotics were amoxicillin (five patients, 35.7%), amoxicillin–clavulanic acid (seven patients, 50%), and cephalosporins (two patients, 14.3%). Patients with immediate-reading positive IDT had a higher rate of positive OPT than those without (p < 0.05, by Fisher’s exact test). The overall sensitivity determined for immediate-reading IDT was 50%, while the specificity was 91.5%. Delayed-reading IDT were negative in all the 96 tested patients, including the 14 patients with positive OPT. Regarding in vitro tests, LTT were performed in nine patients with a positive OPT and 16 negative controls selected among the 82 patients with a negative OPT from the original study. By defining a SI of 2.5 or above for positivity (6), no LTT was positive in patients with positive OPT and two were false positive in patients with a negative OPT to BL (Fig. 1a). However we found an overall higher cell proliferation in children with a confirmed allergy to amoxicillin, suggesting a potential role of T cells in the pathogenesis of non-immediate reactions to BL (Fig. 1b) (p < 0.05, by Mann–Whitney– Wilcoxon test). In addition, BAT were performed in the 14 patients with a positive OPT and 16 negative controls from the original study. According to our criteria, BAT were negative in all tested patients and controls. Overdiagnosis of BL allergy is important and mainly due to the lack of accurate diagnostic test. Although IDT have been used since a long time to diagnose non-immediate BL allergy, their real diagnostic value is not well defined, in previous pediatric studies (2–4, 8, 9), mainly due to the lack of using a gold standard, such as the OPT in patients with positive IDT. Abbreviations: DPTs, drugprovocation tests; IDT, intradermal tests; LTT, lymphocyte transformation tests; BAT, basophil activation test.

Gut T cell receptor-γδ+ intraepithelial lymphocytes are activated selectively by cholera toxin to break oral tolerance in mice

The gut immune system is usually tolerant to harmless foreign antigens such as food proteins. However, tolerance breakdown may occur and lead to food allergy. To study mechanisms underlying food allergy, animal models have been developed in mice by using cholera toxin (CT) to break tolerance. In this study, we identify T cell receptor (TCR)‐γδ+ intraepithelial lymphocytes (IELs) as major targets of CT to break tolerance to food allergens. TCR‐γδ+ IEL‐enriched cell populations isolated from mice fed with CT and transferred to naive mice hamper tolerization to the food allergen β‐lactoglobulin (BLG) in recipient mice which produce anti‐BLG immunoglobulin (Ig)G1 antibodies. Furthermore, adoptive transfer of TCR‐γδ+ cells from CT‐fed mice triggers the production of anti‐CT IgG1 antibodies in recipient mice that were never exposed to CT, suggesting antigen‐presenting cell (APC)‐like functions of TCR‐γδ+ IELs. In contrast to TCR‐αβ+ cells, TCR‐γδ+ IELs bind and internalize CT both in vitro and in vivo. CT‐activated TCR‐γδ+ IELs express major histocompatibility complex (MHC) class II molecules, CD80 and CD86 demonstrating an APC phenotype. CT‐activated TCR‐γδ+ IELs migrate to the lamina propria, where they produce interleukin (IL)‐10 and IL‐17. These results provide in‐vivo evidence for a major role of TCR‐γδ+ IELs in the modulation of oral tolerance in the pathogenesis of food allergy.

Food allergy in mice is modulated through the thymic stromal lymphopoietin pathway

BackgroundThymic stromal lymphopoietin (TSLP) is involved in the pathogenesis of allergic reactions in the skin and the lung. Nevertheless, data on the role of TSLP in food allergy are scarce. We explored the role of TSLP in a mouse model with oral sensitization and oral challenge eliciting food allergy.MethodsTSLP receptor (TSLPR)−/− mice and wild type mice were orally sensitized to β-lactoglobulin in presence of cholera toxin (CT) or CT alone. The elicited immune response was characterized in vitro and the mice were subsequently challenged with the antigen. Lymphocytes from various locations in the gut were activated either by the antigen or by CT and assayed for cytokine secretion.ResultsHere we report that TSLPR−/− are less prone to generate food-induced reactions in conjunction with a decreased antigen-specific IgG1, but not IgE response. In addition, mesenteric lymphnode lymphocytes of TSLPR−/− mice were secreting lower quantities of IL-4, IL-5 and IL-10 after in vivo Ag activation, whereas higher numbers of IL-17 secreting cells were observed. Similarly, activation by the Th2-type adjuvant cholera toxin resulted in an increased frequency of IL-12 and IL-17 secreting lamina propria and mesenteric lymphocytes, together with increased production of IL-12 by activated dendritic cells in TSLPR−/− mice.ConclusionsTSLP can be considered as an essential, but not exclusive, mediator for elicitation of food allergy in mice, as well as a potential target for future therapeutic interventions.

The role of IL-10 in preventing food-induced anaphylaxis

Background: Food allergy is a common condition resulting in a much impaired quality of life. So far, no clearly effective preventive and therapeutic strategies have been established. However, several options have been tested with promising results. Objective: This review examines the potential of various strategies involving an IL-10-mediated effect for tolerance induction to food antigens, mostly for preventing food allergy. Methods: In addition to a review of the literature, we describe and comment on experiments involving a Lactoccocus lactis strain transfected to secrete murine IL-10 directly into the gut. Results/conclusion: This strain was efficient at preventing subsequent sensitization with a common food allergen. There appears to be a potential for such strategies for the prevention of human food allergy but further investigations will be needed to explore them.

The farming environment protects mice from allergen-induced skin contact hypersensitivity

Being born and raised in a farm provides a long‐lasting protection for allergies. The microbial environment provided by farm animals is crucial to induce this protective effect, although underlying immune mechanisms remain elusive.

Effects of an Interleukin-15 Antagonist on Systemic and Skeletal Alterations in Mice with DSS-Induced Colitis

Inflammatory bowel diseases are commonly complicated by weight and bone loss. We hypothesized that IL-15, a pro-inflammatory cytokine expressed in colitis and an osteoclastogenic factor, could play a central role in systemic and skeletal complications of inflammatory bowel diseases. We evaluated the effects of an IL-15 antagonist, CRB-15, in mice with chronic colitis induced by oral 2% dextran sulfate sodium for 1 week, followed by another 1% for 2 weeks. During the last 2 weeks, mice were treated daily with CRB-15 or an IgG2a control antibody. Intestinal inflammation, disease severity, and bone parameters were evaluated at days 14 and 21. CRB-15 improved survival, early weight loss, and colitis clinical score, although colon damage and inflammation were prevented in only half the survivors. CRB-15 also delayed loss of femur bone mineral density and trabecular microarchitecture. Bone loss was characterized by decreased bone formation, but increased bone marrow osteoclast progenitors and osteoclast numbers on bone surfaces. CRB-15 prevented the suppression of osteoblastic markers of bone formation, and reduced osteoclast progenitors at day 14, but not later. However, by day 21, CRB-15 decreased tumor necrosis factor α and increased IL-10 expression in bone, paralleling a reduction of osteoclasts. These results delineate the role of IL-15 on the systemic and skeletal manifestations of chronic colitis and provide a proof-of-concept for future therapeutic developments.