Complexin in ivermectin resistance in body lice.

Ivermectin has emerged as very promising pediculicide, particularly in cases of resistance to commonly used pediculicides. Recently, however, the first field-evolved ivermectin-resistance in lice was reported. To gain insight into the mechanisms underlying ivermectin-resistance, we both looked for mutations in the ivermectin-target site (GluCl) and searched the entire proteome for potential new loci involved in resistance from laboratory susceptible and ivermectin-selected resistant body lice. Polymorphism analysis of cDNA GluCl showed no non-silent mutations. Proteomic analysis identified 22 differentially regulated proteins, of which 13 were upregulated and 9 were downregulated in the resistant strain. We evaluated the correlation between mRNA and protein levels by qRT-PCR and found that the trend in transcriptional variation was consistent with the proteomic changes. Among differentially expressed proteins, a complexin i.e. a neuronal protein which plays a key role in regulating neurotransmitter release, was shown to be the most significantly down-expressed in the ivermectin-resistant lice. Moreover, DNA-mutation analysis revealed that some complexin transcripts from resistant lice gained a premature stop codon, suggesting that this down-expression might be due, in part, to secondary effects of a nonsense mutation inside the gene. We further confirmed the association between complexin and ivermectin-resistance by RNA-interfering and found that knocking down the complexin expression induces resistance to ivermectin in susceptible lice. Our results provide evidence that complexin plays a significant role in regulating ivermectin resistance in body lice and represents the first evidence that links complexin to insecticide resistance.

Placental macrophages are impaired in chorioamnionitis, an infectious pathology of the placenta.

Pregnancy is dependent on maternal-fetal tolerance that may be compromised because of infections or inflammation of the placenta. In this study, we examined whether the context of placental immune tolerance affected the functions of resident macrophages and if their functions were altered during chorioamnionitis, an infectious pathology of the placenta. Macrophages from at-term placentas expressed CD14, exhibited macrophage microbicidal functions, but were less inflammatory than monocyte-derived macrophages. Moreover, placental macrophages spontaneously matured into multinucleated giant cells (MGCs), a property not exhibited by monocyte-derived macrophages, and we detected MGCs of myeloid origin in placental tissue. Compared with placental macrophages, MGCs exhibited a specific phenotype and gene expression signature, consisting of increased cytoskeleton-associated gene expression along with depressed expression of inflammatory response genes. Furthermore, placental macrophages from patients with chorioamnionitis were unable to form MGCs, but this defect was partially corrected by incubating these placental macrophages with control trophoblast supernatants. MGCs formation likely serves to regulate their inflammatory and cytocidal activities in a context that imposes semiallograft acceptance and defense against pathogens.

Chronic hepatitis E virus infection is specifically associated with an interferon-related transcriptional program.

BACKGROUND: Hepatitis E virus (HEV) is a new causative agent of chronic hepatitis in solid organ transplant recipients. Clinical studies suggest that the occurrence and persistence of chronic HEV infection are related to the immunological status of patients. METHODS: We used whole-genome microarray and quantitative reverse transcription polymerase chain reaction (qRT-PCR) to compare the transcriptional profiles of whole blood from 8 kidney transplant recipients with chronic HEV infection and 8 matched kidney transplant recipients without HEV infection. RESULTS: We found that 30 genes in HEV-infected patients were upregulated, compared with those in control patients, as determined by microarray analysis. In contrast, no genes were downregulated. The 30 upregulated genes included 25 interferon-stimulated genes. Increased expression of the genes that encode IFIT1, IFI44L, RSAD2, EPSTI1, and ISG15 was confirmed by qRT-PCR. Interestingly, the expression levels of these genes were associated with the persistence of HEV infection. CONCLUSIONS: Increased expression of interferon-stimulated genes may favor the persistence of an HEV infection. Whether the expression of interferon-stimulated genes is a marker of ongoing viremia or independent prognostic marker of HEV clearance needs further investigations. CLINICAL TRIALS REGISTRATION: NCT01090232.

Monocyte responses in the context of Q fever: from a static polarized model to a kinetic model of activation.

BACKGROUND: Q fever is caused by Coxiella burnetii, a bacterium that persists in M2-polarized macrophages. We wondered whether the concept of M1/M2 polarization is applicable to Q fever patients. METHODS: Monocytes from healthy controls were cultured with IFN-γ and IL-4, agonists of M1 and M2 macrophages, respectively, and their gene expression was assessed using whole-genome microarrays. Selected biomarkers were assessed in blood from Q fever patients by real-time reverse transcription polymerase chain reaction (RT-PCR). RESULTS: Monocytes exhibited early (6-hour) patterns of activation specific to IFN-γ or IL-4 and a late (18-hour) pattern of common activation. Because these responses were not reducible to M1/M2 polarization, we selected biomarkers and tested their relevance in Q fever patients. The early genes NLRC5, RTP4, and RHOH, which were modulated in response to IFN-γ, were up-regulated in patients with acute Q fever, and the expression levels of the late genes ALOX15, CLECSF1, CCL13, and CCL23 were specifically increased in patients with Q fever endocarditis. The RHOH and ALOX15 genes were associated with the activity of acute Q fever and Q fever endocarditis, respectively. CONCLUSIONS: Our results show that the kinetic model of monocyte activation enables a dynamic approach for the evaluation of Q fever patients.

Pathogenic brucellae replicate in human trophoblasts.

Brucellae replicate in a vacuole derived from the endoplasmic reticulum (ER) in epithelial cells, macrophages, and dendritic cells. In animals, trophoblasts are also key cellular targets where brucellae efficiently replicate in association with the ER. Therefore, we investigated the ability of Brucella spp. to infect human trophoblasts using both immortalized and primary trophoblasts. Brucella extensively proliferated within different subpopulations of trophoblasts, suggesting that they constitute an important niche in cases where the fetal-maternal barrier is breached. In extravillous trophoblasts (EVTs), B. abortus and B. suis replicated within single-membrane acidic lysosomal membrane-associated protein 1-positive inclusions, whereas B. melitensis replicated in the ER-derived compartment. Furthermore, B. melitensis but not B. abortus nor B. suis interfered with the invasive capacity of EVT-like cells in vitro. Because EVTs are essential for implantation during early stages of pregnancy, the nature of the replication niche may have a central role during Brucella-associated abortion in infected women.

Low frequency of Vγ9Vδ2 T-cells predicts poor survival in newly diagnosed acute myeloid leukemia.

In several tumor subtypes, increased infiltration of Vγ9Vδ2 T-cells has been shown to have the highest prognostic value compared to other immune subsets. In acute myeloid leukemia (AML), similar findings have been based solely on the inference of transcriptomic data and have not been assessed with respect to confounding factors. This study aimed at determining, by immunophenotypic analysis (flow or mass cytometry) of peripheral blood from AML patients at diagnosis, the prognostic impact of Vγ9Vδ2 T-cell frequency. This was adjusted for potential confounders (age at diagnosis, disease status, European LeukemiaNet classification, leukocytosis, and allogeneic hematopoietic stem cell transplantation as a time-dependent covariate). The cohort was composed of 198 newly diagnosed AML patients. By univariate analysis, patients with lower Vγ9Vδ2 T-cells at diagnosis had significantly lower 5-year overall and relapse-free survivals. These results were confirmed in multivariate analysis (Hazard Ratio [HR]=1.55[1.04-2.30], p=0.030 and HR=1.64[1.06, 2.53], p=0.025). Immunophenotypic alterations observed in patients with lower Vγ9Vδ2 T-cells included a loss of some cytotoxic Vγ9Vδ2 T-cell subsets and a decreased expression of BTN3A on the surface of blasts. Samples expanded regardless of their Vγ9Vδ2 T-cell levels and displayed similar effector functions in vitro. This study confirms the prognostic value of elevated Vγ9Vδ2 T-cells among lymphocytes, in newly diagnosed AML patients. These results provide a strong rationale to consider consolidation protocols aiming at enhancing Vγ9Vδ2 T-cell responses.

Orientia tsutsugamushi, the causative agent of scrub typhus, induces an inflammatory program in human macrophages.

Scrub typhus is a life-threatening disease caused by Orientia tsutsugamushi, a bacterium that primarily infects endothelial cells both in vitro and in vivo. Evidence suggests that the interaction of O. tsutsugamushi with myeloid cells may play a pivotal role in O. tsutsugamushi infection. We demonstrated that O. tsutsugamushi replicated within human monocyte-derived macrophages. Bacteria stimulated the expression of a large number of genes, including type I interferon genes, interferon-stimulated genes, inflammation-associated genes and apoptosis-related genes, and the release of inflammatory cytokines such as Tumor Necrosis Factor and interleukin-1ß. In addition, O. tsutsugamushi induced an M1-type genetic program in macrophages. O. tsutsugamushi viability was required for the type I interferon response and, to a lesser degree, for the inflammatory response. As interferon-γ is known to elicit M1 polarization, we assessed the effect of interferon-γ on the fate of O. tsutsugamushi in macrophages. Exogenous interferon-γ partially inhibited O. tsutsugamushi replication within macrophages. Our results suggest that the inflammatory response induced by O. tsutsugamushi may account for the local and systemic inflammation observed in scrub typhus.

Prognostic Immune Effector Signature in Adult Acute Lymphoblastic Leukemia Patients Is Dominated by γδ T Cells.

The success of immunotherapy has highlighted the critical role of the immune microenvironment in acute lymphoblastic leukemia (ALL); however, the immune landscape in ALL remains incompletely understood and most studies have focused on conventional T cells or NK cells. This study investigated the prognostic impact of circulating γδ T-cell alterations using high-dimensional analysis in a cohort of newly diagnosed adult ALL patients (10 B-ALL; 9 Philadelphia+ ALL; 9 T-ALL). Our analysis revealed common alterations in CD8+ T cells and γδ T cells of relapsed patients, including accumulation of early stage differentiation and increased expression of BTLA and CD73. We demonstrated that the circulating γδ T-cell signature was the most discriminating between relapsed and disease-free groups. In addition, Vδ2 T-cell alterations strongly discriminated patients by relapse status. Taken together, these data highlight the role of ɣδ T cells in adult ALL patients, among whom Vδ2 T cells may be a pivotal contributor to T-cell immunity in ALL. Our findings provide a strong rationale for further monitoring and potentiating Vδ2 T cells in ALL, including in the autologous setting.

CD25high Effector Regulatory T Cells Hamper Responses to PD-1 Blockade in Triple-Negative Breast Cancer.

Regulatory T cells (Treg) impede effective antitumor immunity. However, the role of Tregs in the clinical outcomes of patients with triple-negative breast cancer (TNBC) remains controversial. Here, we found that an immunosuppressive TNBC microenvironment is marked by an imbalance between effector αßCD8+ T cells and Tregs harboring hallmarks of highly suppressive effector Tregs (eTreg). Intratumoral eTregs strongly expressed PD-1 and persisted in patients with TNBC resistant to PD-1 blockade. Importantly, CD25 was the most selective surface marker of eTregs in primary TNBC and metastases compared with other candidate targets for eTreg depletion currently being evaluated in trials for patients with advanced TNBC. In a syngeneic TNBC model, the use of Fc-optimized, IL2 sparing, anti-CD25 antibodies synergized with PD-1 blockade to promote systemic antitumor immunity and durable tumor growth control by increasing effector αßCD8+ T-cell/Treg ratios in tumors and in the periphery. Together, this study provides the rationale for the clinical translation of anti-CD25 therapy to improve PD-1 blockade responses in patients with TNBC. SIGNIFICANCE: An imbalance between effector CD8+ T cells and CD25high effector Tregs marks immunosuppressive microenvironments in αPD-1-resistant TNBC and can be reversed through effector Treg depletion to increase αPD-1 efficacy.

An evaluation of the effects of Lactobacillus ingluviei on body weight, the intestinal microbiome and metabolism in mice.

BACKGROUND: Food can modify the intestinal flora, and Lactobacillus ingluviei has been shown to cause weight gain in chicks and ducks but not in mammals. METHODOLOGY: Female BALB/c mice were divided into a control and two experimental groups and were inoculated either once or twice with L. ingluviei or with PBS. Faecal samples were collected and tested using qPCR in order to detect and quantify Lactobacillus spp., Bacteroidetes spp. and Firmicutes spp. Gene expression was examined in liver and adipose tissue by microarray and qPCR. Metabolic indicators in the plasma were also measured. RESULTS: Mice that were inoculated with 4 × 10(10) L. ingluviei presented a significant increase in weight gain and liver weight and significant increases in Lactobacillus spp. and Firmicutes DNA copy numbers in their faeces. The mRNA levels of fatty acyl synthase (Fas), sterol regulatory element binding factor 1 (Srebp1c), tumour necrosis factor alpha (Tnf), cytochrome P450 2E1 (Cyp2e1), 3-phosphoinositide-dependent protein kinase-1 (Pdpk1), acyl-Coenzyme A dehydrogenase 11 (Acad11), ATP-binding cassette sub family member G (ABCG2) and DEAD box polypeptide 25 (Ddx25) were significantly elevated in the liver tissues of animals in the experimental group. In gonadal adipose tissue, the expression levels of leptin, peroxisome proliferator-activated receptor γ (Pparg) and Srebp1c were significantly higher in animals from the experimental group, whereas the expression of adiponectin was significantly lower in these animals. CONCLUSIONS: The inoculation of L. ingluviei in mice resulted in alterations in the intestinal flora, increased weight gain and liver enlargement, accelerated metabolism and increased inflammation.

High-throughput mass cytometry staining for deep phenotyping of human natural killer cells.

This protocol details the step-by-step procedure for in-depth immune phenotyping of peripheral blood natural killer (NK) cells from clinical samples by mass cytometry. The protocol consists of three main steps: PBMC incubation with a mix of metal-conjugated antibodies for extracellular phenotyping followed by fixation, permeabilization and incubation with a mix of metal-conjugated antibodies for staining of intracellular proteins, and sample acquisition on a mass cytometer. High-dimensional analysis enables the visualization of NK cell subsets and their phenotypical characteristics. For complete details on the use and execution of this protocol, please refer to Chretien et al. (2021).

Placental macrophages: Origin, heterogeneity, function and role in pregnancy-associated infections.

Placental macrophages are a heterogenous population of immune cells present throughout pregnancy. They are essential for maintenance of the homeostatic placenta environment and host defense against infections. The characterization of placental macrophages as well as their activation have been limited for a long time by the lack of convenient tools. The emergence of unbiased methods makes it possible to reappraise the study of placental macrophages. In this review, we discuss the diversity and the functions of placental macrophages to better understand their dysfunctions during placental infections.

Quantification of Immune Variables from Liquid Biopsy in Breast Cancer Patients Links Vδ2+ γδ T Cell Alterations with Lymph Node Invasion.

The rationale for therapeutic targeting of Vδ2+ γδ T cells in breast cancer is strongly supported by in vitro and murine preclinical investigations, characterizing them as potent breast tumor cell killers and source of Th1-related cytokines, backing cytotoxic αß T cells. Nonetheless, insights regarding Vδ2+ γδ T cell phenotypic alterations in human breast cancers are still lacking. This paucity of information is partly due to the challenging scarcity of these cells in surgical specimens. αß T cell phenotypic alterations occurring in the tumor bed are detectable in the periphery and correlate with adverse clinical outcomes. Thus, we sought to determine through an exploratory study whether Vδ2+ γδ T cells phenotypic changes can be detected within breast cancer patients' peripheral blood, along with association with tumor progression. By using mass cytometry, we quantified 130 immune variables from untreated breast cancer patients' peripheral blood. Supervised analyses and dimensionality reduction algorithms evidenced circulating Vδ2+ γδ T cell phenotypic alterations already established at diagnosis. Foremost, terminally differentiated Vδ2+ γδ T cells displaying phenotypes of exhausted senescent T cells associated with lymph node involvement. Thereby, our results support Vδ2+ γδ T cells implication in breast cancer pathogenesis and progression, besides shedding light on liquid biopsies to monitor surrogate markers of tumor-infiltrating Vδ2+ γδ T cell antitumor activity.

Defective Granuloma Formation in Elderly Infected Patients.

Granulomas are compact structures formed in tissues by the immune system in response to aggressions. The in vitro formation of granulomas using circulating mononuclear cells is an innovative method to easily assess the immune response of patients. Monitoring the efficiency of mononuclear cells from patients to form granulomas in vitro would help improve their therapeutic management. Circulating mononuclear cells from 23 elderly patients with sepsis and 24 elderly controls patients were incubated with Sepharose beads coated with either BCG or Coxiella burnetii extracts. The formation of granulomas was measured over 9 days. Most healthy elderly patients (92%) were able to form granulomas in response to BCG and Coxiella burnetii extracts compared to only 48% of infected elderly patients. Undernutrition was significantly associated with impaired granuloma formation in healthy and infected patients. Granulomas typically comprise epithelioid cells and multinucleated giant cells, however, these cells were not detected in samples obtained from patients unable to form granulomas. We also found that the impairment of granuloma formation was associated with reduced production of tumor necrosis factor without overproduction of interleukin-10. Finally, all genes specifically modulated in granulomatous cells were down-modulated in patients with defective granuloma formation. TNFSF10 was the only M1 gene markedly upregulated in patients who did not form granulomas. Our study suggest that defective granuloma formation may be a measurement of altered activation of immune cells which can predispose to nosocomial infections in elderly patients.

Mast Cell Cytonemes as a Defense Mechanism against Coxiella burnetii.

Mast cells (MCs) are critical mediators of inflammation; however, their microbicidal activity against invading pathogens remains largely unknown. Here, we describe a nonpreviously reported antibacterial mechanism used by MCs against Coxiella burnetii, the agent of Q fever. We show that C. burnetii interaction with MCs does not result in bacterial uptake but rather induces the formation of extracellular actin filaments named cytonemes. MC cytonemes express cathelicidin and neutrophil elastase and mediate the capture and destruction of entrapped bacteria. We provide evidence that MC cytoneme formation and microbicidal activity are dependent on the cooperation of the scavenger receptor CD36 and Toll-like receptor 4. Taken together, our results suggest that MCs use an extracellular sophisticated mechanism of defense to eliminate intracellular pathogens, such as C. burnetii, before their entry into host cells.IMPORTANCE Mast cells (MCs) are found in tissues that are in close contact with external environment, such as skin, lungs, or intestinal mucosa but also in the placenta during pregnancy. If their role in mediating allergic conditions is established, several studies now highlight their importance during infection with extracellular pathogens. This study showed a new and effective antimicrobial mechanism of MCs against Coxiella burnetii, an intracellular bacterium whose infection during pregnancy is associated with abortion, preterm labor, and stillbirth. The data reveal that in response to C. burnetii, MCs release extracellular actin filaments that contain antimicrobial agents and are capable to trap and kill bacteria. We show that this mechanism is dependent on the cooperation of two membrane receptors, CD36 and Toll-like receptor 4, and may occur in the placenta during pregnancy by using ex vivo placental MCs. Overall, this study reports an unexpected role for MCs during infection with intracellular bacteria and suggests that MC response to C. burnetii infection is a protective defense mechanism during pregnancy.

A Fast and Reliable Method to Isolate Human Placental Macrophages.

Macrophages are specialized cells involved in recognition, uptake, and destruction of microorganisms. Human placental macrophages are poorly investigated because of the lack of a convenient protocol for their isolation. Here, we present a straightforward and reliable method to isolate macrophages from full-term human placentas. After enzymatic digestion of placental tissue and centrifugation of the cell suspension on a Ficoll cushion, placental macrophages are selected using magnetic beads coated with anti-CD14 antibodies. Isolated cells are characterized by flow cytometry. Ninety eight percent of isolated CD14+ placental macrophages also express the macrophage marker CD68. Thus, this efficient and reliable method yields placental macrophages at high purity and sufficient quantity for functional studies. © 2019 by John Wiley & Sons, Inc.

A transcriptional signature associated with non-Hodgkin lymphoma in the blood of patients with Q fever.

Coxiella burnetii, the agent causing Q fever, has been associated with B-cell non-Hodgkin lymphoma (NHL). To better clarify this link, we analysed the genetic transcriptomic profile of peripheral blood leukocytes from patients with C. burnetii infection to identify possible links to lymphoma. Microarray analyses revealed that 1189 genes were expressed differently (p <.001 and fold change ≥4) in whole blood of patients with C. burnetii infection compared to controls. In addition, 95 genes expressed in patients with non-Hodgkin lymphoma (NHL) and in patients with C. burnetii persistent infection have allowed us to establish the 'C. burnetii-associated NHL signature'. Among these, 33 genes previously found modulated in C. burnetii-associated -NHL by the microarray analysis were selected and their mRNA expression levels were measured in distinct C. burnetii-induced pathologies, namely, acute Q fever, focalized persistent infection, lymphadenitis and C.burnetii-associated NHL. Specific genes involved in anti-apoptotic process were found highly expressed in leukocytes from patients with C. burnetii associated-NHL: MIR17HG, REL and SP100. This signature differed from that found for NHL-control group. Patients with C. burnetii lymphadenitis presented significant elevated levels of BCL2 and ETS1 mRNAs. Altogether, we identified a specific transcriptionnal signature for NHL during C. burnetii infection reflecting the up-regulation of anti-apoptotic processes and the fact that lymphadenitis might constitute a critical step towards lymphomagenesis.

Isolation of Human Placental Mast Cells.

Mast cells have been identified as resident cells of human placental tissue by immunohistological procedures, suggesting that they may play a role in pregnancy. However, the study of placental mast cells requires their isolation. Here, we describe a procedure to isolate placental mast cells from placenta of healthy women. At-term placentas were recovered, and small pieces were excised. After extensive washing, they were digested using enzyme, and cell preparations were centrifuged on a Percoll density gradient. A double positive selection was then performed using magnetic beads covered with CD117 and IgE antibodies. The purity of isolated mast cells was finally analyzed by flow cytometry, and was nearly 90%, demonstrating that our protocol was convenient to obtain fresh placental mast cells in sufficient quantity for research investigations. © 2018 by John Wiley & Sons, Inc.

Cigarette smoke extract interferes with placenta macrophage functions: A new mechanism to compromise placenta functions?

The success of pregnancy depends on the maternal immune system's ability to promote tolerance and host defense. This equilibrium is compromised in inflammatory and infectious impairment of placenta. Smoking during pregnancy exposes the fetus to severe complications which might result from an alteration in placenta macrophages (pMφ) functions. In this study, we assessed the effect of cigarette smoke extract (CSE) on the functions of third trimester pMφs.CSE inhibited particles uptake and the formation of multinucleated giant cells, a recently reported property of pMφs based on their ability to fuse in vitro. These alterations were associated with a CSE-induced abnormal activation of pMφs, which was characterized by an increased release of TNF, interleukin (IL)-33, and decreased IL-6 and IL-10 release. Furthermore, CSE enhanced the expression of metalloproteinase genes known to be involved in tissue remodeling. This effect of CSE on pMφs was specific because CSE affected circulating monocytes in a different way. Finally, we showed that nicotine affected in part the functional properties of pMφs. Taken together, these results showed that CSE modulated the functional activity of pMφs, which may compromise pregnancy.

Immunomodulatory Drugs Exert Anti-Leukemia Effects in Acute Myeloid Leukemia by Direct and Immunostimulatory Activities.

Immunomodulatory drugs (IMiDs) are anticancer drugs with immunomodulatory, anti-angiogenesis, anti-proliferative, and pro-apoptotic properties. IMiDs are currently used for the treatment of multiple myeloma, myelodysplastic syndrome, and B-cell lymphoma; however, little is known about efficacy in acute myeloid leukemia (AML). We proposed in this study to investigate the relevance of IMiDs therapy for AML treatment. We evaluated the effect of IMiDs on primary AML blasts (n = 24), and the impact in natural killer (NK) cell-mediated immunosurveillance of AML. Using primary AML cells and an immunodeficient mouse leukemia xenograft model, we showed that IMiDs induce AML cell death in vitro and impair leukemia progression in vivo. In addition, treatment of AML blasts with IMiDs resulted in enhanced allogeneic NK cell anti-leukemia reactivity. Treatment by pomalidomide of AML blasts enhanced lysis, degranulation, and cytokine production by primary allogeneic NK cells. Furthermore, the treatment with lenalidomide of patients with myeloid malignancies resulted in NK cell phenotypic changes similar to those observed in vitro. IMiDs increased CD56 and decreased NKp30, NKp46, and KIR2D expression on NK cells. Finally, AML blasts treatment with IMiDs induced phenotypic alterations including downregulation of HLA-class I. The effect of pomalidomide was not correlated with cereblon expression and A/G polymorphism in AML cells. Our data revealed, a yet unobserved, dual effects on AML affecting both AML survival and their sensitivity to NK immunotherapy using IMiDs. Our study encourages continuing investigation for the use of IMiDs in AML, especially in combination with conventional therapy or immunotherapy strategies.