Background: Immune checkpoints and their ligands are important actors of lymphocytes and monocytes activation's regulation. Their expression level within T cells changes with aging. Despite the major impact of aging on monocytes, there is no data about the expression of ICs on monocytes from old patients. The objective of our study is to describe the expression of ICs and their ligands on monocytes from young individuals compared to old patients. Methods: We included 18 old control (>75 years old), 10 young control (<55 years old) and 45 old patients with hip fracture (HF). Phenotypical and functional analyses were performed on cryopreserved PBMCs. Results: There is a differential expression of immune checkpoints and their ligands within monocyte subtypes regardless of age at baseline. After stimulation, a differential expression of immune checkpoints in young subjects but not in old subjects was observed which would be in favor of a regulation defect in old subjects. We hypothesize that this lack of regulation could partially explain the excess production of pro-inflammatory cytokines by the stimulated monocytes in old subjects. In HF, we also observe a differential expression of immune checkpoints, especially in old patients with a poor prognosis. Conclusion: Our results suggest that the immune regulation which should take place post-acute stress may be affected in old individuals.

For intradermal (ID) immunisation, novel needle-based delivery systems have been proposed as a better alternative to the Mantoux method. However, the penetration depth of needles in the human skin and its effect on immune cells residing in the different layers of the skin has not been analyzed. A novel and user-friendly silicon microinjection needle (Bella-muTM) has been developed, which allows for a perpendicular injection due to its short needle length (1.4-1.8 mm) and ultrashort bevel. We aimed to characterize the performance of this microinjection needle in the context of the delivery of a particle-based outer mem-brane vesicle (OMV) vaccine using an ex vivo human skin explant model. We compared the needles of 1.4 and 1.8 mm with the conventional Mantoux method to investigate the depth of vaccine injection and the capacity of the skin antigen-presenting cell (APC) to phagocytose the OMVs. The 1.4 mm needle deposited the antigen closer to the epidermis than the 1.8 mm needle or the Mantoux method. Consequently, acti-vation of epidermal Langerhans cells was significantly higher as determined by dendrite shortening. We found that five different subsets of dermal APCs are able to phagocytose the OMV vaccine, irrespective of the device or injection method. ID delivery using the 1.4 mm needle of a OMV-based vaccine allowed epi-dermal and dermal APC targeting, with superior activation of Langerhans cells. This study indicates that the use of a microinjection needle improves the delivery of vaccines in the human skin.(c) 2023 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http:// creativecommons.org/licenses/by/4.0/).

The skin plays a crucial role in host defences against microbial attack and the innate cells must provide the immune system with sufficient information to organize these defences. This unique feature makes the skin a promising site for vaccine administration. Although cellular innate immune events during vaccination have been widely studied, initial events remain poorly understood. Our aim is to determine molecular biomarkers of skin innate reaction after intradermal (i.d.) immunization. Using an ex vivo human explant model from healthy donors, we investigated by NanoLC-MS/MS analysis and MALDI-MSI imaging, to detect innate molecular events (lipids, metabolites, proteins) few hours after i.d. administration of seasonal trivalent influenza vaccine (TIV). This multimodel approach allowed to identify early molecules differentially expressed in dermal and epidermal layers at 4 and 18 h after TIV immunization compared with control PBS. In the dermis, the most relevant network of proteins upregulated were related to cell-to-cell signalling and cell trafficking. The molecular signatures detected were associated with chemokines such as CXCL8, a chemoattractant of neutrophils. In the epidermis, the most relevant networks were associated with activation of antigen-presenting cells and related to CXCL10. Our study proposes a novel step-forward approach to identify biomarkers of skin innate reaction. Significance: To our knowledge, there is no study analyzing innate molecular reaction to vaccines at the site of skin immunization. What is known on skin reaction is based on macroscopic (erythema, redness...), microscopic (epidermal and dermal tissues) and cellular events (inflammatory cell infiltrate). Therefore, we propose a multimodal approach to analyze molecular events at the site of vaccine injection on skin tissue. We identified early molecular networks involved biological functions such cell migration, cell-to-cell interaction and antigen presentation, validated by chemokine expression, in the epidermis and dermis, then could be used as early indicator of success in immunization.

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Background:Our previous work has demonstrated the benefits of transcutaneous immunization in targeting Langerhans cells and preferentially inducing CD8 T-cell responses.Methods:In this randomized phase Ib clinical trial including 20 HIV uninfected volunteers, we compared the safety and immunogenicity of the MVA recombinant vaccine expressing HIV-B antigen (MVA-B) by transcutaneous and intramuscular routes. We hypothesized that the quality of innate and adaptive immunity differs according to the route of immunization and explored the quality of the vector vaccine-induced immune responses. We also investigated the early blood transcriptome and serum cytokine levels to identify innate events correlated with the strength and quality of adaptive immunity.Results:We demonstrate that MVA-B vaccine is safe by both routes, but that the quality and intensity of both innate and adaptive immunity differ significantly. Transcutaneous vaccination promoted CD8 responses in the absence of antibodies and slightly affected gene expression, involving mainly genes associated with metabolic pathways. Intramuscular vaccination, on the other hand, drove robust changes in the expression of genes involved in IL-6 and interferon signalling pathways, mainly those associated with humoral responses, and also some levels of CD8 response.Conclusion:Thus, vaccine delivery route perturbs early innate responses that shape the quality of adaptive immunity.Clinical Trial Registration:http://ClinicalTrials.gov, identifier PER-073-13.

The skin as the interface with the external environment plays a crucial role in host defense against microbial attack. Using this unique feature, skin has become a promising site for vaccine administration. When adaptive immunity is well known, early innate molecular events are poorly understood. We aim to determine novel biomarkers of skin reaction following intradermal (i.d.) immunization. Ex vivo human explant model from healthy donors was used to investigate by NanoLC-MS/MS and MALDI imaging analysis, the early innate molecular events produced at the i.d. Influenza trivalent vaccine (TIV) inoculation site. In this proof-of-concept study, we identified early innate immune proteins differentially expressed in dermal and epidermal layer at 4 to 18 hours following immunization compared to baseline. Up-regulated top network proteins in the dermis were related to cell-to-cell signaling and cell trafficking, whereas in the epidermis, they were associated with antigen-presenting cells at early time point following immunization. In the dermis, CXCL8 chemokines signature was detected following dermal immunization. In the epidermis, CXCL10 linked network proteins was confirmed by qPCR as markers of antigen-presenting cell activation. In addition, skin MALDI imaging allowed to localize in the tissue major metabolites related to these signatures. Our study proposes novel step-forward approach for the identification of biomarkers during early innate reaction at the site of immunization that could correlate with immune protection

Background:Aside from cases of backwash ileitis, the ileal mucosa of patients with ulcerative colitis (UC), an idiotypic inflammatory bowel disease, has received little attention despite the fact that colitis is known to trigger alterations in morphology and/or functions of the small intestine remotely. Methods:The ileal mucosa was studied in patients with UC and in a spontaneous model of colitis (Il10/Nox1dKO mice) mimicking the histological and clinical features of UC and was also studied in acute and chronic murine models of chemically induced colitis. Proliferation and apoptosis were assessed using morphological and immunohistological methods and Western blot analysis. Peyer's patch immune cell subsets were analyzed. Cytokines levels were quantified using quantitative PCR and Luminex xMAP technology. Total RNA from isolated ileal crypts was used for whole genome transcriptome analysis. Results:The most striking features were an increased ileal crypt length associated with an enhanced cell proliferation of the transit-amplifying cells along with activation of the Wnt/&bgr;-catenin and MAPkinase pathways. These changes did not result from intestinal inflammation as assessed by histology and/or pro-inflammatory cytokine expression levels. The increased proliferation rate was dependent on the duration but not on the severity of colitis and was observed in different mouse models of colitis, including the Il10/Nox1dKO model and 2,4,6-trinitrobenzenesulfonic acid–treated mice. Interestingly, the ileal mucosa of patients with UC also displayed longer crypts and enhanced cell proliferation compared with control patients. Conclusions:These data show that despite the absence of inflammation in the small intestine, alterations in the ileal mucosa homeostasis are present in UC.

Ulcerative colitis (UC) is a chronic inflammatory bowel disease affecting the rectum which progressively extents. Its etiology remains unknown and the number of treatments available is limited. Studies of UC patients have identified an unbalanced endoplasmic reticulum (ER) stress in the non-inflamed colonic mucosa. Animal models with impaired ER stress are sensitive to intestinal inflammation, suggesting that an unbalanced ER stress could cause inflammation. However, there are no ER stress-regulating strategies proposed in the management of UC partly because of the lack of relevant preclinical model mimicking the disease. Here we generated the IL10/Nox1dKO mouse model which combines immune dysfunction (IL-10 deficiency) and abnormal epithelium (NADPH oxidase 1 (Nox1) deficiency) and spontaneously develops a UC-like phenotype with similar complications (colorectal cancer) than UC. Our data identified an unanticipated combined role of IL10 and Nox1 in the fine-tuning of ER stress responses in goblet cells. As in humans, the ER stress was unbalanced in mice with decreased eIF2α phosphorylation preceding inflammation. In IL10/Nox1dKO mice, salubrinal preserved eIF2α phosphorylation through inhibition of the regulatory subunit of the protein phosphatase 1 PP1R15A/GADD34 and prevented colitis. Thus, this new experimental model highlighted the central role of epithelial ER stress abnormalities in the development of colitis and defined the defective eIF2α pathway as a key pathophysiological target for UC. Therefore, specific regulators able to restore the defective eIF2α pathway could lead to the molecular remission needed to treat UC.

Background: Recent advances underline intestinal inflammation, induced by handling of the gut during surgery, as crucial mechanism in the pathophysiology of postoperative ileus (POI).Macrophages, in the muscularis layer, and mast cells are the key players in the induction of this inflammatory process.Spleen tyrosine kinase (Syk) is an important kinase involved in macrophage activation as well as in mast cell degranulation and therefore inhibition of Syk pathway may represent an interesting therapeutic approach for POI.In the current study, we have evaluated the effect of the Syk-inhibitor GSK143 as potential treatment to shorten POI.Methods: The effect of an oral single dose (3mg/kg, 1.5 hours before surgery) was evaluated in a mouse model of POI, by analyzing gastrointestinal transit and intestinal muscularis inflammation.The in vitro effect of GSK143 (1 and 3 μM) was evaluated on cultured peritoneal mast cells (pMCs) and bone marrow derived macrophages (BMDMs) stimulated with immune complexes with IgE-anti-TNP (40 ng/mL) and LPS (100 ng/ml) respectively.Results: Administration of GSK143 (3 mg/kg) resulted in a serum level of 0.38±0.24μM 1.5 hours after oral delivery.Treatment with a single dose of GSK143 (3 mg/kg) significantly improved gastrointestinal transit after surgical intestinal manipulated mice compared to placebo (Geometric Center (GC): Placebo, 4.2 ±0.4 vs GSK143, GC; 6.9±0.6).In addition, GSK143 was able to reduce the number of recruited myeloperoxidase (MPO) positive cells (placebo, 187±30 vs GSK143, 44±8, per 0.5 mm2), neutrophils (placebo, 4.6x10-5±0.2x10-5 vs GSK143, 1.7x10-5 ±0.7 x10-5) and monocytes (placebo, 1.6x10-6±0.4x10-6 vs GSK143, 0.8x10-6 ±0.25 x10-6) in the muscularis externa compared to placebo.To define the possible target cells affected, GSK143 was tested in vitro on pMCs and BMDMs.Thirty minutes after GSK143 (1 and 3 μM) or vehicle treatment, pMCs degranulation, quantified by assessment of β-hexosaminidase, was induced by immune complexes with IgE-anti-TNP.Interestingly, GSK143 significantly reduced pMCs degranulation compared to vehicle in a dose dependent manner (GSK143 1μM, 62±7.5% and GSK143 3μM, 76±3.3%inhibition vs vehicle).In line, pre-treatment of BMDMs with GSK143 (1 and 3 μM) prior to LPS challenge significantly lowered the expression of IL-6 (21±4% to 31± 3 % inhibition vs vehicle ), TNF α (39±0.8% to 54±0.7% inhibition vs vehicle ), IL1 β (31±1.3% to 39±1.06% inhibition vs vehicle) and CCL2 (33±5.3% to 43±7% inhibition vs vehicle).Conclusion: Pre-treatment with the Syk-inhibitor GSK143 attenuates intestinal inflammation and consequently restores gastrointestinal transit in a model of POI.In vitro data suggests that both macrophages and mast cells are targeted by GSK143.These findings strongly suggest that Syk inhibition may be a new therapeutical tool to shorten POI.

Intestinal barrier function requires intricate cooperation between intestinal epithelial cells and immune cells. Enteropathogens are able to invade the intestinal lymphoid tissue known as Peyer's patches (PPs) and disrupt the integrity of the intestinal barrier. However, the underlying molecular mechanisms of this process are poorly understood. In mice infected with Yersinia pseudotuberculosis, we found that PP barrier dysfunction is dependent on the Yersinia virulence plasmid and the expression of TLR-2 by hematopoietic cells, but not by intestinal epithelial cells. Upon TLR-2 stimulation, Y. pseudotuberculosis-infected monocytes activated caspase-1 and produced IL-1β. In turn, IL-1β increased NF-κB and myosin light chain kinase activation in intestinal epithelial cells, thus disrupting the intestinal barrier by opening the tight junctions. Therefore, Y. pseudotuberculosis subverts intestinal barrier function by altering the interplay between immune and epithelial cells during infection.

BACKGROUND & AIMS:Ulcerative colitis (UC) is a chronic inflammatory disorder that affects the colonic epithelium. Epidemiology studies indicate an environmental component is involved in pathogenesis, although the primary changes in the digestive epithelium that cause an uncontrolled inflammatory response are not known. Animal studies have shown that altered endoplasmic reticulum (ER) stress response initiates intestinal inflammation in epithelial tissues, but abnormalities associated with ER stress have not been identified in patients with UC.METHODS:Using immunoblotting, real-time polymerase chain reaction, immunohistochemistry, and immunofluorescence analyses, we assessed ER stress signaling in uninflammed colonic mucosa from patients with UC and controls. Genome-wide microarray analysis of actively translated polysome-bound messenger RNA was performed using samples of unaffected mucosa from patients with UC, and data were compared with those from controls.RESULTS:Inositol-requiring kinase and activating transcription factor signaling pathways were activated in inactive colonic epithelium from patients with UC; these mediate proinflammatory and regenerative responses. Blocking phosphorylation of the translation initiation factor 2 (eIF2α), which mediates the integrated stress response, deregulated initiation of translation and reduced the numbers of stress granules in colonic epithelial cells from patients with UC. Genome-wide microarray analysis of actively translated, polysome-bound messenger RNA from patients revealed changes in protein translation that altered colonic epithelial barrier function (levels of detoxification and antioxidant enzymes and proteins that regulate the cell cycle, cell-cell adhesion, and secretion), compared with controls.CONCLUSIONS:Colonic mucosa samples from patients with UC have defects in the eIF2α pathway that controls protein translation and the cell stress response. This pathway might be investigated to identify new therapeutic targets for patients with UC.

Ischemia/reperfusion injury (IRI) causes inflammation and cell injury as a result of activating innate immune signaling. Toll-like receptor 4 (TLR4) has a key role in mediating kidney damages during IRI, but the downstream signaling pathway(s) stimulating apoptosis remains debated. In this study we show that TLR4 mediates MyD88-dependent activation of TNF receptor-associated factor 2, apoptosis signal-regulating kinase 1 (ASK1), and Jun N-terminal kinase (JNK) and p38 MAP kinases in ischemic-reperfused kidneys and posthypoxic renal tubule epithelial cells (RTECs). Hypoxia stimulated the expression of the endoplasmic-resident gp96, which co-immunoprecipitated TLR4, whereas silencing gp96 mRNA expression impaired hypoxia-induced apoptosis in TLR4-expressing RTECs. NAD(P)H oxidase 4 (NOX4) was shown to interact with TLR4 and to be required in lipopolysaccharide-induced production of reactive oxygen species (ROS). IRI stimulated the expression of a 28-kDa NOX4 spliced isoform abundantly expressed in wild-type RTECs, which co-immunoprecipitated with TLR4, but not with gp96 in TLR4-deficient RTECs. Silencing NOX4 mRNA expression impaired hypoxia-induced activation of ASK1 and both JNK and p38, leading to the inhibition of ROS production and apoptosis in posthypoxic TLR4-expressing RTECs. These findings show that, concomitantly to the activation of p38, the gp96/TLR4 interaction is required for activation of ASK1/JNK signaling in posthypoxic mouse RTECs, and that the 28-kDa NOX4 has a key role in TLR4-mediated apoptosis during renal IRI.

The homeostatic self-renewal of the colonic epithelium requires coordinated regulation of the canonical Wnt/beta-catenin and Notch signaling pathways to control proliferation and lineage commitment of multipotent stem cells. However, the molecular mechanisms by which the Wnt/beta-catenin and Notch1 pathways interplay in controlling cell proliferation and fate in the colon are poorly understood. Here we show that NADPH oxidase 1 (NOX1), a reactive oxygen species (ROS)-producing oxidase that is highly expressed in colonic epithelial cells, is a pivotal determinant of cell proliferation and fate that integrates Wnt/beta-catenin and Notch1 signals. NOX1-deficient mice reveal a massive conversion of progenitor cells into postmitotic goblet cells at the cost of colonocytes due to the concerted repression of phosphatidylinositol 3-kinase (PI3K)/AKT/Wnt/beta-catenin and Notch1 signaling. This conversion correlates with the following: (i) the redox-dependent activation of the dual phosphatase PTEN, causing the inactivation of the Wnt pathway effector beta-catenin, and (ii) the downregulation of Notch1 signaling that provokes derepression of mouse atonal homolog 1 (Math1) expression. We conclude that NOX1 controls the balance between goblet and absorptive cell types in the colon by coordinately modulating PI3K/AKT/Wnt/beta-catenin and Notch1 signaling. This finding provides the molecular basis for the role of NOX1 in cell proliferation and postmitotic differentiation.