CXCR4 signaling controls dendritic cell location and activation at steady state and in inflammation.

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.

[The cutaneous virome: from virology to personalized medicine]. / Le virome cutané : de la virologie à la médecine personnalisée.

The virome of the skin, defined as all viruses detected in the skin, represents a significant part of the microbiota. A much more recent discovery than the bacterial flora, the existence of the cutaneous virome has been revealed by recent metagenomic studies. The normal human skin virome is dominated by bacteriophages, Papillomaviridae, whose genomic diversity has proved extraordinary, and Polyomaviridae. Many yet unknown viral genomes within this virome await identification. The composition of the virome of the skin has been shown to be strictly individual and relatively stable over time, resulting from adaptation to everyone's genetics, lifestyle and mechanisms of immunological tolerance finely selected over the course of evolution. Yet little studied, the virome of the skin and all its interactions with other microbiota and the host are attracting growing interest. Indeed, constitutional or acquired alterations in the homeostasis between the commensal virome and the skin, ranging from sub-clinical viral dysbiosis to severe transformation of keratinocytes or adnexal cells, have been observed. These recent observations are stimulating the search for innovative solutions aimed at measuring or even modulating its pathological expression, with a view to personalized medicine.

Neutrophil extracellular traps downregulate lipopolysaccharide-induced activation of monocyte-derived dendritic cells.

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.

Afa/Dr diffusely adhering Escherichia coli strain C1845 induces neutrophil extracellular traps that kill bacteria and damage human enterocyte-like cells.

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.

Case Report: Altered NK Cell Compartment and Reduced CXCR4 Chemotactic Response of B Lymphocytes in an Immunodeficient Patient With HPV-Related Disease.

The study of inborn errors of immunity (IEI) provides unique opportunities to elucidate the microbiome and pathogenic mechanisms related to severe viral infection. Several immunological and genetic anomalies may contribute to the susceptibility to develop Human Papillomavirus (HPV) pathogenesis. They include different acquired immunodeficiencies, EVER1-2 or CIB1 mutations underlying epidermodysplasia verruciformis (EV) syndrome and multiple IEI. Whereas EV syndrome patients are specifically unable to control infections with beta HPV, individuals with IEI show broader infectious and immune phenotypes. The WHIM (warts, hypogammaglobulinemia, infection, and myelokathexis) syndrome caused by gain-of-CXCR4-function mutation manifests by HPV-induced extensive cutaneous warts but also anogenital lesions that eventually progress to dysplasia. Here we report alterations of B and NK cells in a female patient suffering from cutaneous and mucosal HPV-induced lesions due to an as-yet unidentified genetic defect. Despite no detected mutations in CXCR4, B but not NK cells displayed a defective CXCR4-dependent chemotactic response toward CXCL12. In addition, NK cells showed an abnormal distribution with an expanded CD56bright cell subset and defective cytotoxicity of CD56dim cells. Our observations extend the clinical and immunological spectrum of IEI associated with selective susceptibility toward HPV pathogenesis, thus providing new insight on the immune control of HPV infection and potential host susceptibility factors.

The Proteolytic Inactivation of Protein Z-Dependent Protease Inhibitor by Neutrophil Elastase Might Promote the Procoagulant Activity of Neutrophil Extracellular Traps in Sepsis.

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.

Oxidative stress mediates a reduced expression of the activating receptor NKG2D in NK cells from end-stage renal disease patients.

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. CD56(dim) cells, which comprise the majority of NK cells in the periphery, were more affected in this regard than were CD56(bright) 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.

Afa/Dr-expressing, diffusely adhering Escherichia coli strain C1845 triggers F1845 fimbria-dependent phosphatidylserine externalization on neutrophil-like differentiated PLB-985 cells through an apoptosis-independent mechanism.

The enterovirulent Escherichia coli strains potentially involved in inflammatory bowel diseases include diffusely adherent strains expressing Afa/Dr fimbriae (Afa/Dr DAEC). We have previously observed type 1 pilus-mediated interleukin-8 (IL-8) hyperproduction in infected neutrophils. As pathogen induction of host cell death programs and clearance of apoptotic infected cells are crucial for innate immune system homeostasis and host integrity, we examined modulation of neutrophil cell death by Afa/Dr DAEC. Using the human PLB-985 cell line differentiated into fully mature neutrophils, we found that the wild-type enterovirulent E. coli strain C1845 and the recombinant strain DH5alpha/pF1845 (expressing the fimbrial adhesin F1845) similarly induced time-dependent phosphatidylserine (PS) externalization, suggesting a major specific role of this virulence factor. Using small interfering RNA (siRNA) decay-accelerating factor (DAF)-transfected PLB-985 cells, we then showed that this PS externalization was triggered in part by glycosylphosphatidylinositol (GPI)-anchored DAF receptor engagement (leading to tyrosine kinase and protein kinase C activation) and that it required cytoskeleton and lipid raft architectural integrity. PS externalization under these conditions was not dependent on caspases, mitochondria, lysosomes, or reactive oxygen or nitrogen species. F1845-mediated PS externalization was sufficient to enable macrophage engulfment of infected differentiated PLB-985 cells. These findings provide new insights into the neutrophil response to Afa/Dr DAEC infection and highlight a new role for F1845 fimbriae. Interestingly, although apoptosis pathways were not engaged, C1845-infected PLB-985 cells displayed enhanced removal by macrophages, a process that may participate in the resolution of Afa/Dr DAEC infection and related inflammation.

TLR7/8 agonists impair monocyte-derived dendritic cell differentiation and maturation.

Pathogen recognition by TLR activates the innate immune response and is typically followed by the development of an adaptive immune response initiated by antigen presentation. Dendritic cells (DC) are the most efficient APC and express diverse TLRs, including TLR7 and -8, which have been recently identified as targets for ssRNA recognition during viral infection. We have studied the effect of TLR7/8 agonists on DC differentiation and maturation from human monocytes. The synthetic agonist Resiquimod (R-848) or the physiological agonist ssRNA impaired monocyte differentiation to DC phenotypically and functionally. Induced expression of the nonclassical MHC molecules of the CD1 family in DC was inhibited at the protein and mRNA levels, and antigen acquisition was inhibited. Proinflammatory cytokine (including IL-6, IL-8, TNF-alpha, IL-1beta) and IL-10 production were induced during DC differentiation. Cross-talk between TLR4 and TLR7/8 was revealed as immature DC, which had been differentiated in the presence of R-848 were insensitive to LPS-mediated maturation and cytokine production but still induced allostimulation. These data lead us to suggest that ongoing viral activation of TLR7/8 could alter the adaptive immune response by modifying DC differentiation and by down-regulating DC responsiveness to a subsequent bacterial TLR4-mediated signal.

Design of protease-resistant myelin basic protein-derived peptides by cleavage site directed amino acid substitutions.

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.

Obstructive Sleep Apnoea Modulates Airway Inflammation and Remodelling in Severe Asthma.

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<0.05). RBM thickness and OSA severity assessed by the AHI were negatively correlated (rho = -0.65, p<0.05). OSA and non-OSA patients did not differ in age, sex, BMI, lung function, asthma control findings or treatment. CONCLUSION: Mild OSA in patients with severe asthma is associated with increased proportion of neutrophils in sputum and changes in airway remodelling.

Neutropenic Mice Provide Insight into the Role of Skin-Infiltrating Neutrophils in the Host Protective Immunity against Filarial Infective Larvae.

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.

Altered chemotactic response to CXCL12 in patients carrying GATA2 mutations.

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 CD56(bright) 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.

An improved strategy to recover large fragments of functional human neutrophil extracellular traps.

Netosis is a recently described neutrophil function that leads to the release of neutrophil extracellular traps (NETs) in response to various stimuli. NETs are filaments of decondensed chromatin associated with granular proteins. In addition to their role against microorganisms, NETs have been implicated in autoimmunity, thrombosis, and tissue injury. Access to a standardized source of isolated NETs is needed to better analyze the roles of NETs. The aim of this study was to develop a procedure yielding soluble, well-characterized NET preparations from fresh human neutrophils. The calcium ionophore A23187 was chosen to induce netosis, and the restriction enzyme AluI was used to prepare large NET fragments. DNA and proteins were detected by electrophoresis and specific labeling. Some NET proteins [histone 3, lactoferrin (LF)] were quantified by western blotting, and double-stranded DNA (dsDNA) was quantified by immunofluorescence. Co-existence of dsDNA and neutrophil proteins confirmed the quality of the NET preparations. Their biological activity was checked by measuring elastase (ELA) activity and bacterial killing against various strains. Interindividual differences in histone 3, LF, ELA, and dsDNA relative contents were observed in isolated NETs. However, the reproducibility of NET preparation and characterization was validated, suggesting that this interindividual variability was rather related to donor variation than to technical bias. This standardized protocol is suitable for producing, isolating, and quantifying functional NETs that could serve as a tool for studying NET effects on immune cells and tissues.

Chemotherapeutic agents targeting the tubulin cytoskeleton modify LPS-induced cytokine secretion by dendritic cells and increase antigen presentation.

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.

Dendritic cells differentiated in the presence of a single-stranded viral RNA sequence conserve their ability to activate CD4 T lymphocytes but lose their capacity for Th1 polarization.

Monocyte-derived dendritic cells (DCs) differentiate in the presence of Toll-like-receptor (TLR) ligands in the course of ongoing infections. A single-stranded RNA (ssRNA) sequence, corresponding to the sequence of the U5 region of human immunodeficiency virus type 1 RNA, was used to mimic viral activation of TLR7 in human DCs. We determined the effector potential of DCs differentiated in the presence of this ssRNA molecule (ssRNA-DCs). ssRNA-DCs phenotypically resembled mature DCs. In contrast, their capacity to allostimulate naive CD4(+) T cells resembled that of conventional immature DCs and could be increased by TLR4 stimulation. Th1 polarization of CD4(+) T cells and production of interleukin 12p70 (IL-12p70) by ssRNA-DCs were selectively abrogated in response to a late TLR4, but not in response to a CD40, maturation signal. Inhibition of p38 mitogen-activated protein kinase partially restored IL-12p70 secretion but did not restore Th1 polarization, whereas addition of exogenous IL-12 led to recovery of Th1 polarization. In contrast to lipopolysaccharide, ssRNA induced IL-12p70 production at the very earliest stages of DC differentiation, indicating a particular role for TLR7 in monocyte-derived DCs recently engaged in differentiation. These data demonstrate generation of phenotypically mature DCs with the ability to expand CD4(+) T lymphocytes lacking Th1/2-polarizing capacity.

Contrasting cytoskeletal regulation of MHC class II peptide presentation by human B cells or dendritic cells.

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.