Recent advances in epicutaneous immunotherapy and potential applications in food allergy.

Given the potent immunological properties of the skin, epicutaneous immunotherapy (EPIT) emerges as a promising treatment approach for inducing immune tolerance, particularly for food allergies. Targeting the highly immunocompetent, non-vascularized epidermis allows for the application of microgram amounts of allergen while significantly reducing the risk of allergen passage into the bloodstream, thus limiting systemic allergen exposure and distribution. This makes EPIT highly suitable for the treatment of potentially life-threatening allergies such as food allergies. Multiple approaches to EPIT are currently under investigation for the treatment of food allergy, and these include the use of allergen-coated microneedles, application of allergen on the skin pretreated by tape stripping, abrasion or laser-mediated microperforation, or the application of allergen on the intact skin using an occlusive epicutaneous system. To date, the most clinically advanced approach to EPIT is the Viaskin technology platform. Viaskin is an occlusive epicutaneous system (patch) containing dried native allergen extracts, without adjuvants, which relies on frequent application for the progressive passage of small amounts of allergen to the epidermis through occlusion of the intact skin. Numerous preclinical studies of Viaskin have demonstrated that this particular approach to EPIT can induce potent and long-lasting T-regulatory cells with broad homing capabilities, which can exert their suppressive effects in multiple organs and ameliorate immune responses from different routes of allergen exposure. Clinical trials of the Viaskin patch have studied the efficacy and safety for the treatment of life-threatening allergies in younger patients, at an age when allergic diseases start to occur. Moreover, this treatment approach is designed to provide a non-invasive therapy with no restrictions on daily activities. Taken together, the preclinical and clinical data on the use of EPIT support the continued investigation of this therapeutic approach to provide improved treatment options for patients with allergic disorders in the near future.

Epicutaneous allergen immunotherapy induces a profound and selective modulation in skin dendritic-cell subsets.

BACKGROUND: Epicutaneous immunotherapy (EPIT) protocols have recently been developed to restore tolerance in patients with food allergy. The mechanisms by which EPIT protocols promote desensitization rely on a profound immune deviation of pathogenic T- and B-cell responses. OBJECTIVE: To date, little is known about the contribution of skin dendritic cells (skDCs) to T-cell remodeling and EPIT efficacy. METHODS: We capitalized on a preclinical model of food allergy to ovalbumin (OVA) to characterize the phenotype and functions of OVA+ skDCs throughout the course of EPIT. RESULTS: Our results showed that both Langerhans cells and dermal conventional cDC1 and cDC2 subsets retained their ability to capture OVA in the skin and to migrate toward the skin-draining lymph nodes during EPIT. However, their activation/maturation status was significantly impaired, as evidenced by the gradual and selective reduction of CD86, CD40, and OVA protein expression in respective subsets. Phenotypic changes during EPIT were also characterized by a progressive diversification of single-cell gene signatures within each DC subset. Interestingly, we observed that OVA+ Langerhans cells progressively lost their capacity to prime CD4+ TEFF cells, but gained regulatory T-cell stimulatory properties. In contrast, cDC1 were inefficient in priming CD4+ TEFF cells or in reactivating TMEM cells in vitro, whereas cDC2 retained moderate stimulatory properties, and progressively biased type 2 immunity toward type 1 and type 17 responses. CONCLUSIONS: Our results therefore emphasize that the acquisition of distinct phenotypic and functional specializations by skDCs during EPIT is at the cornerstone of the desensitization process.

Epicutaneous immunotherapy protects cashew-sensitized mice from anaphylaxis.

BACKGROUND: The prevalence of tree nut allergy has increased worldwide, and cashew has become one of the most common food allergens. More critically, cashew allergy is frequently associated with severe anaphylaxis. Despite the high medical need, no approved treatment is available and strict avoidance and preparedness for prompt treatment of allergic reactions are considered dual standard of care. In the meantime, Phase III study results suggest investigational epicutaneous immunotherapy (EPIT) may be a relevant and safe treatment for peanut allergy and may improve the quality of life for many peanut allergic children. OBJECTIVE: We aimed to evaluate the capacity of EPIT to provide protection against cashew-induced anaphylaxis in a relevant mouse model. METHODS: The efficacy of EPIT was evaluated by applying patches containing cashew allergens to cashew-sensitized mice. As negative control, sham mice received patches containing excipient. Following treatment, mice were challenged orally to cashew and anaphylactic symptoms, as well as plasmatic levels of mast-cell proteases (mMCP)-1/7, were quantified. RESULTS: Of 16 weeks of EPIT significantly protects against anaphylaxis by promoting a faster recovery of challenged mice. This protection was characterized by a significant reduction of temperature drop and clinical symptoms, 60 minutes after challenge. This was associated with a decrease in mast-cell reactivity as attested by the reduction of mMCP-1/7 in plasma, suggesting that EPIT specifically decrease IgE-mediated anaphylaxis. CONCLUSION: We demonstrate that EPIT markedly reduced IgE-mediated allergic reactions in a mouse model of cashew allergy, which suggests that EPIT may be a relevant approach to treating cashew allergy.

Antigen Uptake by Langerhans Cells Is Required for the Induction of Regulatory T Cells and the Acquisition of Tolerance During Epicutaneous Immunotherapy in OVA-Sensitized Mice.

The skin is a major immunologic organ that may induce protection, sensitization or tolerance. Epicutaneous immunotherapy (EPIT) has been proposed as an attractive strategy to actively treat food allergy and has been shown to induce tolerance in sensitized mice through the induction of Foxp3+ regulatory T cells (Tregs), especially CD62L+ Tregs. Among immune cells in the skin, dendritic cells are key players in antigen-specific immune activation or regulation. The role of different populations of skin DCs in tolerance induction remains to be elucidated. Using OVA-sensitized BALB/c mice, we demonstrated that the application of a patch containing OVA-A647 to the skin resulted in allergen uptake by Langerhans cells (LCs) and CD11b+ dermal cDC2 and subsequent migration into skin draining lymph nodes. These 2 populations induced Foxp3 expression in CD4+ cells in vitro. Only LCs induced LAP+ cells and CD62L+ Tregs. Using Langerin-eGFP-DTR mice, we analyzed the role of LCs in the mechanisms of tolerance induction by EPIT in vivo. Following complete depletion of LCs, a dramatic decrease in the number of OVA+ DCs and OVA+ CD11b+ dermal cDC2 was observed in skin draining lymph nodes 48 h after epicutaneous application. Likewise, 2 weeks of EPIT in non-depleted mice induced Foxp3+ Tregs, especially CD62L+, and LAP+ Tregs in skin draining lymph nodes and spleen, whereas no induction of Tregs was observed in LC-depleted mice. Following 8 weeks of treatment, EPIT-treated mice showed significant protection against anaphylaxis accompanied by a significant increase of Foxp3+ Tregs, especially CD62L+ Tregs, which was not seen in the absence of LCs. In summary, although both LCs and CD11b+ dermal cDC2s could induce regulatory T cells, the absence of LCs during EPIT impaired treatment efficacy, indicating their crucial role in skin-induced tolerance.

Differences in phenotype, homing properties and suppressive activities of regulatory T cells induced by epicutaneous, oral or sublingual immunotherapy in mice sensitized to peanut.

Allergen-specific immunotherapy has been proposed as an attractive strategy to actively treat food allergy using the following three different immunotherapy routes: oral (OIT), sublingual (SLIT) and epicutaneous (EPIT) immunotherapy. Regulatory T cells (Tregs) have been shown to have a pivotal role in the mechanisms of immunotherapy. The aim of this study was to compare the phenotype and function of Tregs induced in peanut-sensitized BALB/c mice using these three routes of treatment. We show that although EPIT, OIT and SLIT were all able to effectively desensitize peanut-sensitized mice, they induced different subsets of Tregs. Foxp3+ Tregs were induced by the three treatment routes but with greater numbers induced by EPIT. EPIT and OIT also increased the level of LAP+ Tregs, whereas SLIT induced IL-10+ cells. The suppressive activity of EPIT-induced Tregs did not depend on IL-10 but required CTLA-4, whereas OIT acted through both mechanisms and SLIT was strictly dependent on IL-10. Moreover, the three routes influenced the homing properties of induced Tregs differently, with a larger repertoire of chemokine receptors expressed by EPIT-induced Tregs compared with OIT- and SLIT- induced cells, resulting in different protective consequences against allergen exposure. Furthermore, whereas OIT- or SLIT-induced Tregs lost their suppressive activities after treatment was discontinued, the suppressive activities of EPIT-induced Tregs were still effective 8 weeks after the end of treatment, suggesting the induction of a more long-lasting tolerance. In summary, EPIT, OIT and SLIT mediated desensitization through the induction of different subsets of Tregs, leading to important differences in the subsequent protection against allergen exposure and the possible induction of tolerance.

Epicutaneous immunotherapy for food allergy as a novel pathway for oral tolerance induction.

Epicutaneous immunotherapy is a developing technique, aiming at desensitizing patients with food allergy with less risks that oral ingestion or injection could generate. Several clinical trials have been performed and are currently running, in milk and peanut allergy, assessing the safety of the technique and its efficacy. Preclinical models indicate a major role in the mechanisms of desensitization, for example, Tregs and epigenetic modifications.

Specific epicutaneous immunotherapy prevents sensitization to new allergens in a murine model.

BACKGROUND: Allergy to cow's milk increases the risk of sensitization to other foods in young children. OBJECTIVES: We sought to evaluate the effect of early epicutaneous immunotherapy (EPIT) on further sensitization to peanut or house dust mite (HDM) in a murine model of sensitization to cow's milk. METHODS: BALB/c mice orally sensitized to milk were epicutaneously treated with a Viaskin patch (DBV Technologies) loaded with milk proteins for 8 weeks. Mice were then sensitized to peanut or HDM. After sensitization to peanut, mice were exposed to a peanut regimen known to induce eosinophilic esophageal inflammation. After sensitization to HDM, mice were challenged with aerosols to HDM, and airway hyperresponsiveness was evaluated by using plethysmography. Humoral response was also analyzed. The role of regulatory T (Treg) cells was evaluated by adoptively transferring Treg cells from milk EPIT-treated mice to naive mice before sensitization to peanut. Protection against anaphylaxis was also investigated. Methylation of the promoter region of transcription factors was analyzed by using PCR assays. RESULTS: In milk-sensitized mice specific EPIT prevented further sensitization to peanut or HDM. EPIT significantly modified the humoral response, reduced TH2 cytokine levels, decreased eosinophilic esophageal infiltration, and suppressed airway hyperresponsiveness. The protective effect was sustained over 2 months. Moreover, the adoptive transfer of milk EPIT Treg cells completely prevented sensitization to peanut and peanut-induced anaphylaxis. Milk EPIT enhanced methylation of the GATA-3 promoter region. CONCLUSIONS: Our results showed that EPIT influences the natural history of allergy and reduces the risk of further sensitization through a Treg cell-dependent mechanism.

Intact skin and not stripped skin is crucial for the safety and efficacy of peanut epicutaneous immunotherapy (EPIT) in mice.

BACKGROUND: Epicutaneous immunotherapy (EPIT) on intact skin with an epicutaneous delivery system has already been used in preclinical and clinical studies. In epicutaneous vaccination and immunotherapy, the stripping of skin before application of the allergen is suggested to facilitate the passage of allergen through immune cells. OBJECTIVES: The aim of this study was to compare the immunological response induced by EPIT performed on intact and stripped skin in a mouse model of peanut allergy. METHODS: After oral sensitization with peanut and cholera toxin, BALB/c mice were epicutaneously treated using an epicutaneous delivery system (Viaskin® (DBV Technologies, Paris) applied either on intact skin or on stripped skin. Following EPIT, mice received an exclusive oral peanut regimen, aimed at triggering esophageal and jejunal lesions. We assessed eosinophil infiltration by histology, mRNA expression in the esophagus, antibody levels and peripheral T-cell response. RESULTS: EPIT on intact skin significantly reduced Th2 immunological response (IgE response and splenocyte secretion of Th2 cytokines) as well as esophageal eosinophilia (2.7 ± 0.9, compared to Sham 19.9 ± 1.5, p < 0.01), mRNA expression of Th2 cytokines in tissue and intestinal villus sub-atrophia (2.9 ± 0.2 vs Sham, 2.1 ± 0.2, p < 0.05). By contrast, EPIT on stripped skin reinforced Th2 systemic immunological response as well as eosinophil infiltration (26.8 ± 15.1), mRNA expression of Th2 cytokines and duodenal villus/crypt-ratio (2.4 ± 0.3). CONCLUSIONS: Epicutaneous allergen-specific immunotherapy needs the integrity of superficial layers of the stratum corneum to warranty safety of treatment and to induce a tolerogenic profile of the immune response.

Epicutaneous immunotherapy (EPIT) blocks the allergic esophago-gastro-enteropathy induced by sustained oral exposure to peanuts in sensitized mice.

BACKGROUND: Food allergy may affect the gastrointestinal tract and eosinophilia is often associated with allergic gastrointestinal disorders. Allergy to peanuts is a life-threatening condition and effective and safe treatments still need to be developed. The present study aimed to evaluate the effects of sustained oral exposure to peanuts on the esophageal and jejunal mucosa in sensitized mice. We also evaluated the effects of desensitization with epicutaneous immunotherapy (EPIT) on these processes. METHODS: Mice were sensitized by gavages with whole peanut protein extract (PPE) given with cholera toxin. Sensitized mice were subsequently exposed to peanuts via a specific regimen and were then analysed for eosinophilia in the esophagus and gut. We also assessed mRNA expression in the esophagus, antibody levels, and peripheral T-cell response. The effects of EPIT were tested when intercalated with sensitization and sustained oral peanut exposure. RESULTS: Sustained oral exposure to peanuts in sensitized mice led to severe esophageal eosinophilia and intestinal villus sub-atrophia, i.e. significantly increased influx of eosinophils into the esophageal mucosa (136 eosinophils/mm(2)) and reduced villus/crypt ratios (1.6±0.15). In the sera, specific IgE levels significantly increased as did secretion of Th2 cytokines by peanut-reactivated splenocytes. EPIT of sensitized mice significantly reduced Th2 immunological response (IgE response and splenocyte secretion of Th2 cytokines) as well as esophageal eosinophilia (50 eosinophils/mm(2), p<0.05), mRNA expression of Th2 cytokines in tissue--eotaxin (p<0.05), IL-5 (p<0.05), and IL-13 (p<0.05)--GATA-3 (p<0.05), and intestinal villus sub-atrophia (2.3±0.15). EPIT also increased specific IgG2a (p<0.05) and mRNA expression of Foxp3 (p<0.05) in the esophageal mucosa. CONCLUSIONS: Gastro-intestinal lesions induced by sustained oral exposure in sensitized mice are efficaciously treated by allergen specific EPIT.

Epicutaneous Immunotherapy Compared with Sublingual Immunotherapy in Mice Sensitized to Pollen (Phleum pratense).

Background. The aim of this study was to compare the efficacy of epicutaneous immunotherapy (EPIT) to sublingual immunotherapy (SLIT) in a model of mice sensitized to Phleum pratense pollen. Methods. BALB/c mice were sensitized by sub-cutaneous route to pollen protein extract mixed treated for 8 weeks, using sham, EPIT, or SLIT. Measurements involved the serological response and cytokine profile from reactivated splenocytes, plethysmography after aerosol challenge to pollen, cell, and cytokine contents in the bronchoalveolar lavages (BALs). Results. After immunotherapy, sIgE was significantly decreased in the treated groups compared to sham (P < 0.001), whereas sIgG2a increased with EPIT and SLIT (P < 0.001 and P < 0.005 versus sham). Reactivated splenocytes secreted higher levels of Th2 cytokines with sham (P < 0.01). Penh values were higher in sham than EPIT and SLIT. Eosinophil recruitment in BAL was significantly reduced only by EPIT (P < 0.01). Conclusion. In this model of mice sensitized to pollen, EPIT was at least as efficient as SLIT.

Human peritoneal mesothelial cells respond to bacterial ligands through a specific subset of Toll-like receptors.

BACKGROUND: Bacterial infection remains a major cause of morbidity and mortality in peritoneal dialysis (PD) patients worldwide. Previous studies have identified a key role for mesothelial cells, lining the peritoneal cavity, in coordinating inflammation and host defense. Toll-like receptor (TLR) involvement in early activation events within the mesothelium, however, remains poorly defined. To investigate the initiation of bacterial peritonitis, we characterized TLR activation by bacterial ligands in human peritoneal mesothelial cells (HPMC). METHODS: Primary HPMC were isolated from omental biopsies and TLR expression detected by real-time polymerase chain reaction (PCR), reverse transcription (RT)-PCR and flow cytometry. The responsiveness of HPMC to specific bacterial TLR agonists was determined using chemokine production as a biological readout. The requirement for CD14 in HPMC responses to a clinically relevant Staphylococcus epidermidis cell-free supernatant (SES) was investigated using soluble CD14 or anti-CD14-blocking antibodies. RESULTS: Real-time PCR detected TLR1-6 messenger RNA expression in HPMC and responses to TLR2/1 and TLR2/6 ligands and SES. No cell surface TLR4 expression or responses to lipopolysaccharide were detectable in HPMC, but they did respond to flagellin, a TLR5 ligand. SES-mediated responses were dependent on TLR2 but did not require CD14 in HPMC for optimal efficiency, unlike peripheral blood mononuclear cells. HPMC expression of TLR2 was also modulated by TLR2 ligands and inflammatory cytokines. CONCLUSIONS: These data suggest that mesothelial cell activation by TLR2/1, TLR2/6 and TLR5 contributes to bacterial recognition influencing the course of the infective process and has implications for improving treatment of infection in PD patients.

Epicutaneous immunotherapy results in rapid allergen uptake by dendritic cells through intact skin and downregulates the allergen-specific response in sensitized mice.

Epicutaneous immunotherapy onto intact skin has proved to be an efficient and safe alternative treatment of allergy in an animal model with various allergens and in children for cow's milk allergy. The aim of this study was to analyze the different steps of the immunological handling of the allergen when deposited on intact skin using an epicutaneous delivery system and its immune consequences in sensitized BALB/c mice. As expected, when applied on intact skin, OVA exhibits neither a passive passage through the skin nor any detectable systemic delivery. The current study demonstrates that, after a prolonged application on intact skin, OVA is taken up by dendritic cells in the superficial layers of the stratum corneum and transported, after internalization, to the draining lymph nodes, with variations according to the previous level of sensitization of the mice. When OVA is applied with the epicutaneous delivery system repeatedly, specific local and systemic responses are down-modulated in association with the induction of regulatory T cells. Besides providing new insights into skin function in the presence of allergens, this study indicates that the skin might have a tolerogenic role, at least when kept intact.

Epicutaneous immunotherapy using a new epicutaneous delivery system in mice sensitized to peanuts.

BACKGROUND: Peanut allergy is a life-threatening condition for which new efficient and safe treatment is expected. We evaluated epicutaneous immunotherapy (EPIT) as a new alternative treatment for peanut allergy in sensitized mice. METHODS: Sixty BALB/c mice were sensitized by gavages with peanut protein extract (PPE) mixed with cholera toxin. An epicutaneous delivery system, coated with 100 µg PPE (Viaskin®, DBV Technologies, Paris, France), was applied to intact skin every week during 48 h (EPIT; n = 20). This group was compared with sensitized mice treated with subcutaneous immunotherapy (SCIT; n = 20), untreated sensitized mice (sham, n = 20), and naive mice (naive; n = 20). After the 8-week treatment, a histamine release test, airway hyperreactivity measurement by plethysmography, and a resistance-compliance measurement after the challenge were performed. Blood and bronchoalveolar lavage were sampled for serology, cytokines, and cytology. RESULTS: Specific IgE (sIgE) increased after sensitization in the EPIT (0.26 µg/ml) and SCIT (0.21 µg/ml) groups and decreased after treatment (0.09 µg/ml, p < 0.001 and 0.06 µg/ml, p < 0.001, respectively). The IgG1/IgG2a ratio decreased in the EPIT and SCIT groups versus the sham group (3.7; p < 0.001 and 2.7; p < 0.01 and 15.1, respectively). At the higher metacholine concentration, enhanced pause values were lower in the EPIT and SCIT groups than in the sham group (7.29, 6.74, and 10.99, p < 0.01, respectively), and did not differ from that of the naive group (5.06). Resistance-compliance was reversed in the treated groups versus the sham group (p < 0.001). IL-4, IL-5, IL-13, eotaxin, and eosinophils were reduced in the BAL of the EPIT and SCIT groups versus the sham group (p < 0.001). CONCLUSION: In peanut-sensitized mice, based on biological and physiological responses, EPIT is as efficient as subcutaneous treatment which is the reference method in immunotherapy.

Phosphatidylethanolamine-esterified eicosanoids in the mouse: tissue localization and inflammation-dependent formation in Th-2 disease.

In this study, murine peritoneal macrophages from naïve lavage were found to generate four phospholipids that contain 12-hydroxyeicosatetraenoic acid (12-HETE). They comprise three plasmalogen and one diacyl phosphatidylethanolamines (PEs) (16:0p, 18:1p, 18:0p, and 18:0a at sn-1) and are absent in macrophages from 12/15-lipoxygenase (12/15-LOX)-deficient mice. They are generated acutely in response to calcium mobilization, are primarily cell-associated, and are detected on the outside of the plasma membrane. Levels of 12-HETE-PEs in naïve lavage are in a similar range to those of free 12-HETE (5.5 +/- 0.2 ng or 18.5 +/- 1.03 ng/lavage for esterified versus free, respectively). In healthy mice, 12/15-LOX-derived 12-HETE-PEs are found in the peritoneal cavity, peritoneal membrane, lymph node, and intestine, with a similar distribution to 12/15-LOX-derived 12-HETE. In vivo generation of 12-HETE-PEs occurs in a Th2-dependent model of murine lung inflammation associated with interleukin-4/interleukin-13 expression. In contrast, in Toll receptor-dependent peritonitis mediated either by live bacteria or bacterial products, 12-HETE-PEs are rapidly cleared during the acute phase then reappear during resolution. The human homolog, 18:0a/15-HETE-PE inhibited human monocyte generation of cytokines in response to lipopolysaccharide. In summary, a new family of lipid mediators generated by murine macrophages during Th2 inflammation are identified and structurally characterized. The studies suggest a new paradigm for lipids generated by 12/15-LOX in inflammation involving formation of esterified eicosanoids.

12/15-Lipoxygenase regulates the inflammatory response to bacterial products in vivo.

The peritoneal macrophage (Mphi) is the site of greatest 12/15-lipoxygenase (12/15-LOX) expression in the mouse; however, its immunoregulatory role in this tissue has not been explored. Herein, we show that 12/15-LOX is expressed by 95% of resident peritoneal CD11b(high) cells, with the remaining 5% being 12/15-LOX(-). 12/15-LOX(+) cells are phenotypically defined by high F4/80, SR-A, and Siglec1 expression, and enhanced IL-10 and G-CSF generation. In contrast, 12/15-LOX(-) cells are a dendritic cell population. Resident peritoneal Mphi numbers were significantly increased in 12/15-LOX(-/-) mice, suggesting alterations in migratory trafficking or cell differentiation in vivo. In vitro, Mphi from 12/15-LOX(-/-) mice exhibit multiple abnormalities in the regulation of cytokine/growth factor production both basally and after stimulation with Staphylococcus epidermidis cell-free supernatant. Resident adherent cells from 12/15-LOX(-/-) mice generate more IL-1, IL-3, GM-CSF, and IL-17, but less CCL5/RANTES than do cells from wild-type mice, while Staphylococcus epidermidis cell-free supernatant-elicited 12/15-LOX(-/-) adherent cells release less IL-12p40, IL-12p70, and RANTES, but more GM-CSF. This indicates a selective effect of 12/15-LOX on peritoneal cell cytokine production. In acute sterile peritonitis, 12/15-LOX(+) cells and LOX products were cleared, then reappeared during the resolution phase. The peritoneal lavage of 12/15-LOX(-/-) mice showed elevated TGF-beta1, along with increased immigration of monocytes/Mphi, but decreases in several cytokines including RANTES/CCL5, MCP-1/CCL2, G-CSF, IL-12-p40, IL-17, and TNF-alpha. No changes in neutrophil or lymphocyte numbers were seen. In summary, endogenous 12/15-LOX defines the resident MPhi population and regulates both the recruitment of monocytes/Mphi and cytokine response to bacterial products in vivo.

Oncostatin M receptor-beta signaling limits monocytic cell recruitment in acute inflammation.

Although the IL-6-related cytokine oncostatin M (OSM) affects processes associated with disease progression, the specific function of OSM in the face of an inflammatory challenge remains unclear. In this report, a peritoneal model of acute inflammation was used to define the influence of OSM on chemokine-mediated leukocyte recruitment. When compared with wild-type and IL-6-deficient mice, peritoneal inflammation in oncostatin M receptor-beta-deficient (OSMR-KO) mice resulted in enhanced monocytic cell trafficking. In contrast to IL-6-deficient mice, OSMR-KO mice displayed no difference in neutrophil and lymphocyte migration. Subsequent in vitro studies using human peritoneal mesothelial cells and an in vivo appraisal of inflammatory chemokine expression after peritoneal inflammation identified OSM as a prominent regulator of CCL5 expression. Specifically, OSM inhibited IL-1beta-mediated NF-kappaB activity and CCL5 expression in human mesothelial cells. This was substantiated in vivo where peritoneal inflammation in OSMR-KO mice resulted in a temporal increase in both CCL5 secretion and NF-kappaB activation. These findings suggest that IL-6 and OSM individually affect the profile of leukocyte trafficking, and they point to a hitherto unidentified interplay between OSM signaling and the inflammatory activation of NF-kappaB.

Circulating neutrophils maintain physiological blood pressure by suppressing bacteria and IFNgamma-dependent iNOS expression in the vasculature of healthy mice.

Whether leukocytes exert an influence on vascular function in vivo is not known. Here, genetic and pharmacologic approaches show that the absence of neutrophils leads to acute blood pressure dysregulation. Following neutrophil depletion, systolic blood pressure falls significantly over 3 days (88.0 +/- 3.5 vs 104.0 +/- 2.8 mm Hg, day 3 vs day 0, mean +/- SEM, P < .001), and aortic rings from neutropenic mice do not constrict properly. The constriction defect is corrected using l-nitroarginine-methyl ester (L-NAME) or the specific inducible nitric oxide synthase (iNOS) inhibitor 1400W, while acetylcholine relaxation is normal. iNOS- or IFNgamma-deficient mice are protected from neutropenia-induced hypotension, indicating that iNOS-derived nitric oxide (NO) is responsible and that its induction involves IFNgamma. Oral enrofloxacin partially inhibited hypotension, implicating bacterial products. Roles for cyclooxygenase, complement C5, or endotoxin were excluded, although urinary prostacyclin metabolites were elevated. Neutrophil depletion required complement opsinization, with no evidence for intravascular degranulation. In summary, circulating neutrophils contribute to maintaining physiological tone in the vasculature, at least in part through suppressing early proinflammatory effects of infection. The speed with which hypotension developed provides insight into early changes that occur in the absence of neutrophils and illustrates the importance of constant surveillance of mucosal sites by granulocytes in healthy mice.

Activated platelets and monocytes generate four hydroxyphosphatidylethanolamines via lipoxygenase.

12/15-Lipoxygenase (LOX) mediates immune-regulatory activities not accounted for by its known free acid eicosanoids, suggesting that additional lipids may be generated by activated cells. To characterize novel LOX-derived lipids, a lipidomic approach was utilized. Ionophore-activated interleukin-4-treated human peripheral monocytes generated up to 10-fold more esterified 15-hydroxyeicosatetraenoic acid (15-HETE) than free in a phosphatidylinositol 3-kinase- and protein kinase C-sensitive manner. Precursor scanning electrospray ionization/tandem spectroscopy for m/z 319 (HETE, [M-H](-)) showed 4 ions at m/z 738, 764, 766, and 782 that were identified using tandem spectroscopy and MS3 as specific diacyl and plasmalogen 15-HETE phosphatidylethanolamines. Using H (18)(2)O water, the compounds were shown to form by direct oxidation of endogenous phosphatidylethanolamine (PE) by 15-LOX, with PE being the preferred phospholipid pool containing 15-HETE. Similarly, human platelets generated 4 analogous PE lipids that contained 12-HETE and increased significantly in response to ionophore, collagen, or convulxin. These products were retained in the cells, in contrast to free acids, which are primarily secreted. Precursor scanning of platelet extracts for the major platelet-derived prostanoid, thromboxane B2 (m/z 369.2), did not reveal PE esters, indicating that this modification is restricted to the LOX pathway. In summary, we show formation of PE-esterified HETEs in immune cells that may contribute to LOX signaling in inflammation.

Changes in soluble factor-mediated CD8+ cell-derived antiviral activity in cynomolgus macaques infected with simian immunodeficiency virus SIVmac251: relationship to biological markers of progression.

Cross-sectional studies have shown that the capacity of CD8+ cells from human immunodeficiency virus (HIV)-infected patients and simian immunodeficiency virus (SIV) SIVmac-infected macaques to suppress the replication of human and simian immunodeficiency viruses in vitro depends on the clinical stage of disease, but little is known about changes in this antiviral activity over time in individual HIV-infected patients or SIV-infected macaques. We assessed changes in the soluble factor-mediated noncytolytic antiviral activity of CD8+ cells over time in eight cynomolgus macaques infected with SIVmac251 to determine the pathophysiological role of this activity. CD8+ cell-associated antiviral activity increased rapidly in the first week after viral inoculation and remained detectable during the early phase of infection. The net increase in antiviral activity of CD8+ cells was correlated with plasma viral load throughout the 15 months of follow-up. CD8+ cells gradually lost their antiviral activity over time and acquired virus replication-enhancing capacity. Levels of antiviral activity correlated with CD4+ T-cell counts after viral set point. Concentrations of beta-chemokines and interleukin-16 in CD8+ cell supernatants were not correlated with this antiviral activity, and alpha-defensins were not detected. The soluble factor-mediated antiviral activity of CD8+ cells was neither cytolytic nor restricted to major histocompatibility complex. This longitudinal study strongly suggests that the increase in noncytolytic antiviral activity from baseline and the maintenance of this increase over time in cynomolgus macaques depend on both viral replication and CD4+ T cells.

Kinetics of lymphocyte proliferation during primary immune response in macaques infected with pathogenic simian immunodeficiency virus SIVmac251: preliminary report of the effect of early antiviral therapy.

The aim of this study was to evaluate the kinetics of lymphocyte proliferation during primary infection of macaques with pathogenic simian immunodeficiency virus (SIV) and to study the impact of short-term postexposure highly active antiretroviral therapy (HAART) prophylaxis. Twelve macaques were infected by intravenous route with SIVmac251 and given treatment for 28 days starting 4 h postexposure. Group 1 received a placebo, and groups 2 and 3 received combinations of zidovudine (AZT), lamivudine (3TC), and indinavir. Macaques in group 2 received AZT (4.5 mg/kg of body weight), 3TC (2.5 mg/kg), and indinavir (20 mg/kg) twice per day by the oral route whereas macaques in group 3 were given AZT (4.5 mg/kg) and 3TC (2.5 mg/kg) subcutaneously twice per day, to improve the pharmacokinetic action of these drugs, and a higher dose of indinavir (60 mg/kg). The kinetics of lymphocyte proliferation were analyzed by monitoring 5-bromo-2'-deoxyuridine (BrdU) uptake ex vivo and by fluorescence-activated cell sorting analysis. HAART did not protect against SIV infection but did strongly impact on virus loads: viremia was delayed and lowered during antiviral therapy in group 2, with better control after treatment was stopped, and in group 3, viremia was maintained at lower levels during treatment, with virus even undetectable in the blood of some macaques, but there was no evidence of improved control of the virus after treatment. We provide direct evidence that dividing NK cells are detected earlier than dividing T cells in the blood (mostly in CD45RA(-) T cells), mirroring plasma viremia. Dividing CD8(+) T cells were detected earlier than dividing CD4(+) T cells, and the highest percentages of proliferating T cells coincided with the first evidence of partial control of peak viremia and with an increase in the percentage of circulating gamma interferon-positive CD8(+) T cells. The level of cell proliferation in the blood during SIV primary infection was clearly associated with viral replication levels because the inhibition of viral replication by postexposure HAART strongly reduced lymphocyte proliferation. The results and conclusions in this study are based on experiments in a small numbers of animals and are thus preliminary.