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Dendritic cells (DCs) encompass several cell subsets that collaborate to initiate and regulate immune responses. Proper DC localization determines their function and requires the tightly controlled action of chemokine receptors. All DC subsets express CXCR4, but the genuine contribution of this receptor to their biology has been overlooked. We addressed this question using natural CXCR4 mutants resistant to CXCL12-induced desensitization and harboring a gain of function that cause the warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome (WS), a rare immunodeficiency associated with high susceptibility to the pathogenesis of human papillomavirus (HPV). We report a reduction in the number of circulating plasmacytoid DCs (pDCs) in WHIM patients, whereas that of conventional DCs is preserved. This pattern was reproduced in an original mouse model of WS, enabling us to show that the circulating pDC defect can be corrected upon CXCR4 blockade and that pDC differentiation and function are preserved, despite CXCR4 dysfunction. We further identified proper CXCR4 signaling as a critical checkpoint for Langerhans cell and DC migration from the skin to lymph nodes, with corollary alterations of their activation state and tissue inflammation in a model of HPV-induced dysplasia. Beyond providing new hypotheses to explain the susceptibility of WHIM patients to HPV pathogenesis, this study shows that proper CXCR4 signaling establishes a migration threshold that controls DC egress from CXCL12-containing environments and highlights the critical and subset-specific contribution of CXCR4 signal termination to DC biology.

Septic shock is the archetypal clinical setting in which extensive crosstalk between inflammation and coagulation dysregulates the latter. The main anticoagulant systems are systematically impaired, depleted, and/or downregulated. Protein Z-dependent protease inhibitor (ZPI) is an anticoagulant serpin that not only targets coagulation factors Xa and XIa but also acts as an acute phase reactant whose plasma concentration rises in inflammatory settings. The objective of the present study was to assess the plasma ZPI antigen level in a cohort of patients suffering from septic shock with or without overt-disseminated intravascular coagulation (DIC). The plasma ZPI antigen level was approximately 2.5-fold higher in the patient group (n = 100; 38 with DIC and 62 without) than in healthy controls (n = 31). The elevation's magnitude did not appear to depend on the presence/absence of DIC. Furthermore, Western blots revealed the presence of cleaved ZPI in plasma from patients with severe sepsis, independently of the DIC status. In vitro, ZPI was proteolytically inactivated by purified neutrophil elastase (NE) and by NE on the surface of neutrophil extracellular traps (NETs). The electrophoretic pattern of ZPI after NE-catalyzed proteolysis was very similar to that resulting from the clotting process—suggesting that the cleaved ZPI observed in severe sepsis plasma is devoid of anticoagulant activity. Taken as a whole, our results (1) suggest that NE is involved in ZPI inactivation during sepsis, and (2) reveal a novel putative mechanism for the procoagulant activity of NETs in immunothrombosis.

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Our knowledge and control of the pathogenesis induced by the filariae remain limited due to experimental obstacles presented by parasitic nematode biology and the lack of selective prophylactic or curative drugs. Here we thought to investigate the role of neutrophils in the host innate immune response to the infection caused by the Litomosoides sigmodontis murine model of human filariasis using mice harboring a gain-of-function mutation of the chemokine receptor CXCR4 and characterized by a profound blood neutropenia (Cxcr4+/1013). We provided manifold evidence emphasizing the major role of neutrophils in the control of the early stages of infection occurring in the skin. Firstly, we uncovered that the filarial parasitic success was dramatically decreased in Cxcr4+/1013 mice upon subcutaneous delivery of the infective stages of filariae (infective larvae, L3). This protection was linked to a larger number of neutrophils constitutively present in the skin of the mutant mice herein characterized as compared to wild type (wt) mice. Indeed, the parasitic success in Cxcr4+/1013 mice was normalized either upon depleting neutrophils, including the pool in the skin, or bypassing the skin via the intravenous infection of L3. Second, extending these observations to wt mice we found that subcutaneous delivery of L3 elicited an increase of neutrophils in the skin. Finally, living L3 larvae were able to promote in both wt and mutant mice, an oxidative burst response and the release of neutrophil extracellular traps (NET). This response of neutrophils, which is adapted to the large size of the L3 infective stages, likely directly contributes to the anti-parasitic strategies implemented by the host. Collectively, our results are demonstrating the contribution of neutrophils in early anti-filarial host responses through their capacity to undertake different anti-filarial strategies such as oxidative burst, degranulation and NETosis., Author Summary Filariases are chronic debilitating diseases caused by parasitic nematodes affecting more than 150 million people worldwide. None of the current drugs are selective, neither able to eliminate the parasites nor to prevent new infections once the drug pressure has waned. Therefore, blocking the entry and the migration of the infective larvae (L3) could be an efficient way to control the infection. In the present study we investigated the early interaction between the host and the L. sigmodontis murine filariasis with a focus on the neutrophils in the innate host responses. We uncovered a key role of neutrophils in the control of infection provided by the CXCR4-gain-of-function mice (Cxcr4+/1013) that display a blood neutropenia as well as an accumulation of skin-infiltrating neutrophils. Overall, we reveal that in the early phase of filariasis, i.e. after L3 are delivered into the skin and before they reach their site for reproduction, neutrophils are critical elements of the host innate protective response arsenal. A better understanding of their indirect and/or effector role(s) may provide mechanistic clues to host factors implicated in parasitic nematode entry and potentially lead to the identification of new drug targets.

MHC class II-mediated antigen presentation by B lymphocytes or dendritic cells (DC) initiates CD4+ T lymphocyte activation. In B lymphocytes, MHC class II peptide presentation has been characterised by recruitment of MHC class II, F-actin and lipid rafts to the B cell-T cell immunological synapse. We now show that MHC class II engagement in B lymphocytes induced lipid raft-independent Rho and Rac activation and that inhibition of either Rho-GTPase activation or actin polymerisation in the B cell abrogated T cell activation without altering B cell-T cell conjugate formation. Short-hairpin RNA studies excluded a role for the Cdc42 effector Wiskott-Aldrich syndrome protein. In contrast, antigen presentation by DC was Rho-GTPase-independent although actin was recruited to the DC-T cell interaction site. Moreover, actin depolymerisation in the DC significantly increased T cell activation without altering the number of DC-T cell conjugates. Finally we show that stable recruitment of HLA-DR to the site of the immunological synapse is not a uniform observation in DC and demonstrate reduced HLA-DR expression at the site of microtubule organising centre polarization. Therefore although actin accumulates in DC and B lymphocytes at the immunological synapse with antigen-specific T lymphocytes, this does not reflect comparable functional roles of their actin cytoskeletons in antigen presentation.

GATA2 deficiency—formerly described as MonoMAC syndrome; dendritic cells, monocytes, B cells, and natural killer cell deficiency; familial myelodysplastic syndrome/acute myeloid leukemia; or Emberger syndrome—encompasses a range of hematologic and nonhematologic anomalies, mainly characterized by monocytopenia, B lymphopenia, natural killer cell cytopenia, neutropenia, immunodeficiency, and a high risk of developing acute myeloid leukemia. Herein, we present 7 patients with GATA2 deficiency recruited into the French Severe Chronic Neutropenia Registry, which enrolls patients with all kinds of congenital neutropenia. We performed extended immunophenotyping of their whole blood lymphocyte populations, together with the analysis of their chemotactic responses. Lymphopenia was recorded for B and CD4+ T cells in 6 patients. Although only 3 patients displayed natural killer cell cytopenia, the CD56bright natural killer subpopulation was nearly absent in all 7 patients. Natural killer cells from 6 patients showed decreased CXCL12/CXCR4-dependent chemotaxis, whereas other lymphocytes, and most significantly B lymphocytes, displayed enhanced CXCL12-induced chemotaxis compared with healthy volunteers. Surface expression of CXCR4 was significantly diminished in the patients’ natural killer cells, although the total expression of the receptor was found to be equivalent to that of natural killer cells from healthy individual controls. Together, these data reveal that GATA2 deficiency is associated with impaired membrane expression and chemotactic dysfunctions of CXCR4. These dysfunctions may contribute to the physiopathology of this deficiency by affecting the normal distribution of lymphocytes and thus potentially affecting the susceptibility of patients to associated infections.

Background Obstructive sleep apnoea (OSA) is frequently observed in severe asthma but the causal link between the 2 diseases remains hypothetical. The role of OSA-related systemic and airway neutrophilic inflammation in asthma bronchial inflammation or remodelling has been rarely investigated. The aim of this study was to compare hallmarks of inflammation in induced sputum and features of airway remodelling in bronchial biopsies from adult patients with severe asthma with and without OSA. Materials and Methods An overnight polygraphy was performed in 55 patients referred for difficult-to-treat asthma, who complained of nocturnal respiratory symptoms, poor sleep quality or fatigue. We compared sputum analysis, reticular basement membrane (RBM) thickness, smooth muscle area, vascular density and inflammatory cell infiltration in bronchial biopsies. Results In total, 27/55 patients (49%) had OSA diagnosed by overnight polygraphy. Despite a moderate increase in apnoea-hypopnoea index (AHI; 14.2±1.6 event/h [5–35]), the proportion of sputum neutrophils was higher and that of macrophages lower in OSA than non-OSA patients, with higher levels of interleukin 8 and matrix metalloproteinase 9. The RBM was significantly thinner in OSA than non-OSA patients (5.8±0.4 vs. 7.8±0.4 μm, p

Major histocompatibility complex (MHC) molecules are a key element of the cellular immune response. Encoded by the MHC they are a family of highly polymorphic peptide receptors presenting peptide antigens for the surveillance by T cells. We have shown that certain organic compounds can amplify immune responses by catalyzing the peptide loading of human class II MHC molecules HLA-DR. Here we show now that they achieve this by interacting with a defined binding site of the HLA-DR peptide receptor. Screening of a compound library revealed a set of adamantane derivatives that strongly accelerated the peptide loading rate. The effect was evident only for an allelic subset and strictly correlated with the presence of glycine at the dimorphic position beta86 of the HLA-DR molecule. The residue forms the floor of the conserved pocket P1, located in the peptide binding site of MHC molecule. Apparently, transient occupation of this pocket by the organic compound stabilizes the peptide-receptive conformation permitting rapid antigen loading. This interaction appeared restricted to the larger Gly(beta86) pocket and allowed striking enhancements of T cell responses for antigens presented by these "adamantyl-susceptible" MHC molecules. As catalysts of antigen loading, compounds targeting P1 may be useful molecular tools to amplify the immune response. The observation, however, that the ligand repertoire can be affected through polymorphic sites form the outside may also imply that environmental factors could induce allergic or autoimmune reactions in an allele-selective manner.

The asparagine-specific endoprotease (AEP) controls lysosomal processing of the potential autoantigen myelin basic protein (MBP) by human B lymphoblastoid cells, a feature implicated in the immunopathogenesis of multiple sclerosis. In this study, we demonstrate that freshly isolated human B lymphocytes lack significant AEP activity and that cleavage by AEP is dispensable for proteolytic processing of MBP in this type of cell. Instead, cathepsin (Cat) G, a serine protease that is not endogenously synthesized by B lymphocytes, is internalized from the plasma membrane and present in lysosomes from human B cells where it represents a major functional constituent of the proteolytic machinery. CatG initialized and dominated the destruction of intact MBP by B cell-derived lysosomal extracts, degrading the immunodominant MBP epitope and eliminating both its binding to MHC class II and a MBP-specific T cell response. Degradation of intact MBP by CatG was not restricted to a lysosomal environment, but was also performed by soluble CatG. Thus, the abundant protease CatG might participate in eliminating the immunodominant determinant of MBP. Internalization of exogenous CatG represents a novel mechanism of professional APC to acquire functionally dominant proteolytic activity that complements the panel of endogenous lysosomal enzymes.

Small-molecular compounds with hydrogen bond (H-bond) donor function are able to trigger exchange reactions of MHC class II ligands. Here, we show that their effect is not limited to short peptides. Also encephalitogenic myelin basic protein (MBP) is transferred with great efficiency onto HLA-DR molecules when H-bond donor molecules such as parachlorphenol (pCP) are present. The effect was observed not only with soluble MHC class II but also with HLA-DR1 and HLA-DR2 molecules on the cell surface. The improved loading of APC translates directly into improved T cell activation. In the presence of pCP T cells reacted at significantly lower antigen concentrations, an effect observed with purified MBP protein as well as with crude spinal cord homogenate. The 'accidental' transfer of autoantigens such as MBP onto activated APC might trigger fatal autoimmune reactions and small molecules as catalysts of this process could represent risk factors, which had not been accounted for as yet.

Polymorphonuclear neutrophils (PMN) play a central role in inflammation and participate in its control, notably by modulating dendritic cell (DC) functions via soluble mediators or cell–cell contacts. Neutrophil extracellular traps (NETs) released by PMN could play a role in this context. To evaluate NET effects on DC maturation, we developed a model based on monocyte-derived DC (moDC) and calibrated NETs isolated from fresh human PMN. We found that isolated NETs alone had no discernable effect on moDC. In contrast, they downregulated LPS-induced moDC maturation, as shown by decreased surface expression of HLA-DR, CD80, CD83, and CD86, and by downregulated cytokine production (TNF-α, IL-6, IL-12, IL-23), with no increase in the expression of tolerogenic DC genes. Moreover, the presence of NETs during moDC maturation diminished the capacity of these moDC to induce T lymphocyte proliferation in both autologous and allogeneic conditions, and modulated CD4+ T lymphocyte polarization by promoting the production of Th2 cytokines (IL-5 and IL-13) and reducing that of Th1 and Th17 cytokines (IFN-γ and IL-17). Interestingly, the expression and activities of the lymphoid chemokine receptors CCR7 and CXCR4 on moDC were not altered when moDC matured in the presence of NETs. Together, these findings reveal a new role for NETs in adaptive immune responses, modulating some moDC functions and thereby participating in the control of inflammation.

We recently documented the neutrophil response to enterovirulent diffusely adherent Escherichia coli expressing Afa/Dr fimbriae (Afa/Dr DAEC), using the human myeloid cell line PLB-985 differentiated into fully mature neutrophils. Upon activation, particularly during infections, neutrophils release neutrophil extracellular traps (NETs), composed of a nuclear DNA backbone associated with antimicrobial peptides, histones, and proteases, which entrap and kill pathogens. Here, using fluorescence microscopy and field emission scanning electron microscopy, we observed NET production by PLB-985 cells infected with the Afa/Dr wild-type (WT) E. coli strain C1845. We found that these NETs were able to capture, immobilize, and kill WT C1845 bacteria. We also developed a coculture model of human enterocyte-like Caco-2/TC7 cells and PLB-985 cells previously treated with WT C1845 and found, for the first time, that the F-actin cytoskeleton of enterocyte-like cells is damaged in the presence of bacterium-induced NETs and that this deleterious effect is prevented by inhibition of protease release. These findings provide new insights into the neutrophil response to bacterial infection via the production of bactericidal NETs and suggest that NETs may damage the intestinal epithelium, particularly in situations such as inflammatory bowel diseases.

Recent studies support the idea that certain chemotherapeutic agents do not act only by cytotoxicity, but also have immunomodulatory activity. Because of their role in mitosis chemotherapeutic agents targeting microtubules are widely used, and this study determined the outcome of disturbing tubulin cytoskeleton dynamics on human dendritic cell function. Dendritic cells (DCs) play a major role in the generation of the adaptive immune response owing to their capacity for antigen presentation leading to T lymphocyte activation and differentiation and there is compelling evidence for their contribution to the antitumoral immune response. Two agents that target the tubulin cytoskeleton were used, taxol and colchicine, and a brief pretreatment with either increased antigen presentation by DCs independently of significant phenotypic change, cell death, or cytokine production. Although taxol and colchicine use different mechanisms to disrupt microtubules, NF-kappabeta was activated by either. We therefore determined whether the cytokine secretion profile in response to lipopolysaccharide (LPS) was modified. LPS stimulation of DCs induced IL-10, IL-12p70, TNFalpha and IL-1beta secretion, and taxol pretreatment modified this response by down-regulating IL-1beta secretion whereas colchicine induced a proinflammatory cytokine profile with reduced IL-10 and increased IL-12p70 and TNFalpha secretion. Taken together, these data reveal new immunomodulatory strategies of microtubule disrupting agents in dendritic cells, that of modifying the cytokine response to LPS and that of increasing T lymphocyte activation without induction of inflammatory cytokine secretion by dendritic cells.

To characterize the immune defect of patients with end-stage renal disease (ESRD), we performed NK cell subset analysis in 66 patients with ESRD treated by hemodialysis (n = 59) or peritoneal dialysis (n = 7). Compared with healthy blood donors, patients undergoing chronic dialysis showed a profound decrease in NKG2D+ cells within both the CD8+ T cell (58% vs 67%, p = 0.03) and NK cell (39% vs 56%, p = 0.002) populations. CD56dim cells, which comprise the majority of NK cells in the periphery, were more affected in this regard than were CD56bright cells. Uremic serum could decrease NKG2D expression on NK cells from healthy donors. Among factors that could contribute to the decrease in NKG2D expression in ESRD patients, reactive oxygen species (ROS) play a major role. We found that catalase could reverse the effects of uremic serum on NKG2D expression (p < 0.001) and that ROS down-regulated NKG2D at the mRNA level and at the NK cell surface. Additionally, ESRD patients had both increased membrane-bound MHC class I-related chain A (MICA) on monocytes (p = 0.04) and increased soluble MICA (203 pg/ml vs 110 pg/ml; p < 0.001). Both ROS and uremic serum could significantly increase in vitro the expression of the NKG2D ligand MICA on the renal epithelial cell line HK-2. Taken together, these studies suggest for the first time that both low NKG2D expression and up-regulation of its ligand MICA are related to ROS production and may be involved in the immune deficiency of ESRD patients.

Multiple Sclerosis (MS) is considered to be a T cell-mediated autoimmune disease. An attractive strategy to prevent activation of autoaggressive T cells in MS, is the use of altered peptide ligands (APL), which bind to major histocompatibility complex class II (MHC II) molecules. To be of clinical use, APL must be capable of resisting hostile environments including the proteolytic machinery of antigen presenting cells (APC). The current design of APL relies on cost- and labour-intensive strategies. To overcome these major drawbacks, we used a deductive approach which involved modifying proteolytic cleavage sites in APL. Cleavage site-directed amino acid substitution of the autoantigen myelin basic protein (MBP) resulted in lysosomal protease-resistant, high-affinity binding peptides. In addition, these peptides mitigated T cell activation in a similar fashion as conventional APL. The strategy outlined allows the development of protease-resistant APL and provides a universal design strategy to improve peptide-based immunotherapeutics.