Eric Muraille

Bacterial c-di-GMP is an immunostimulatory molecule.

Cyclic diguanylate (c-di-GMP) is a bacterial intracellular signaling molecule. We have shown that treatment with exogenous c-di-GMP inhibits Staphylococcus aureus infection in a mouse model. We now report that c-di-GMP is an immodulator and immunostimulatory molecule. Intramammary treatment of mice with c-di-GMP 12 and 6 h before S. aureus challenge gave a protective effect and a 10,000-fold reduction in CFUs in tissues (p < 0.001). Intramuscular vaccination of mice with c-di-GMP coinjected with S. aureus clumping factor A (ClfA) Ag produced serum with significantly higher anti-ClfA IgG Ab titers (p < 0.001) compared with ClfA alone. Intraperitoneal injection of mice with c-di-GMP activated monocyte and granulocyte recruitment. Human immature dendritic cells (DCs) cultured in the presence of c-di-GMP showed increased expression of costimulatory molecules CD80/CD86 and maturation marker CD83, increased MHC class II and cytokines and chemokines such as IL-12, IFN-γ, IL-8, MCP-1, IFN-γ-inducible protein 10, and RANTES, and altered expression of chemokine receptors including CCR1, CCR7, and CXCR4. c-di-GMP-matured DCs demonstrated enhanced T cell stimulatory activity. c-di-GMP activated p38 MAPK in human DCs and ERK phosphorylation in human macrophages. c-di-GMP is stable in human serum. We propose that cyclic dinucleotides like c-di-GMP can be used clinically in humans and animals as an immunomodulator, immune enhancer, immunotherapeutic, immunoprophylactic, or vaccine adjuvant.

Revisiting the Th1/Th2 paradigm

The identification of subsets of CD4+ helper cells producing distinct pattern of cytokines has provided a valuable framework for understanding how different effector populations of immune cells can be recruited in vivo during infection. In the view of most investigators, Th1 and Th2 cells produce factors that serve as their own autocrine factors and cytokines exerting suppressive activities on each others development and activity. This concept intuitively explains the natural tendency of immune responses to become progressively polarized. However, several experimental observations appear difficult to rationalize with a simple, ‘symmetrical’ Th1/Th2 paradigm including those that Th1 cells do not produce their own growth factor; that both Th1 and Th2 cells can promote inflammatory responses; that interleukin‐10 (IL‐10) inhibits inflammatory responses in a Th1/Th2‐independent fashion; that IL‐10 promotes the development of Th1‐type effector cells; and that IL‐12 can amplify pre‐established Th2 responses. The purpose of the present analysis is to provide a revised model for better understanding how cytokines regulate immune responses in vivo.

iNOS-producing inflammatory dendritic cells constitute the major infected cell type during the chronic Leishmania major infection phase of C57BL/6 resistant mice.

Leishmania major parasites reside and multiply in late endosomal compartments of host phagocytic cells. Immune control of Leishmania growth absolutely requires expression of inducible Nitric Oxide Synthase (iNOS/NOS2) and subsequent production of NO. Here, we show that CD11b+ CD11c+ Ly-6C+ MHC-II+ cells are the main iNOS-producing cells in the footpad lesion and in the draining lymph node of Leishmania major-infected C57BL/6 mice. These cells are phenotypically similar to iNOS-producing inflammatory DC (iNOS-DC) observed in the mouse models of Listeria monocytogenes and Brucella melitensis infection. The use of DsRed-expressing parasites demonstrated that these iNOS-producing cells are the major infected population in the lesions and the draining lymph nodes. Analysis of various genetically deficient mouse strains revealed the requirement of CCR2 expression for the recruitment of iNOS-DC in the draining lymph nodes, whereas their activation is strongly dependent on CD40, IL-12, IFN-γ and MyD88 molecules with a partial contribution of TNF-α and TLR9. In contrast, STAT-6 deficiency enhanced iNOS-DC recruitment and activation in susceptible BALB/c mice, demonstrating a key role for IL-4 and IL-13 as negative regulators. Taken together, our results suggest that iNOS-DC represent a major class of Th1-regulated effector cell population and constitute the most frequent infected cell type during chronic Leishmania major infection phase of C57BL/6 resistant mice.

MyD88-Dependent Activation of B220−CD11b+LY-6C+ Dendritic Cells during Brucella melitensis Infection

IFN-γ is a key cytokine controlling Brucella infection. One of its major function is the stimulation of Brucella-killing effector mechanisms, such as inducible NO synthase (iNOS)/NOS2 activity, in phagocytic cells. In this study, an attempt to identify the main cellular components of the immune response induced by Brucella melitensis in vivo is made. IFN-γ and iNOS protein were analyzed intracellularly using flow cytometry in chronically infected mice. Although TCRβ+CD4+ cells were the predominant source of IFN-γ in the spleen, we also identified CD11b+LY-6C+LY-6G−MHC-II+ cells as the main iNOS-producing cells in the spleen and the peritoneal cavity. These cells appear similar to inflammatory dendritic cells recently described in the mouse model of Listeria monocytogenes infection and human psoriasis: the TNF/iNOS-producing dendritic cells. Using genetically deficient mice, we demonstrated that the induction of iNOS and IFN-γ-producing cells due to Brucella infection required TLR4 and TLR9 stimulation coupled to Myd88-dependent signaling pathways. The unique role of MyD88 was confirmed by the lack of impact of Toll-IL-1R domain-containing adaptor inducing IFN-β deficiency. The reduction of IFN-γ+ and iNOS+ cell frequency observed in MyD88-, TLR4-, and TLR9-deficient mice correlated with a proportional lack of Brucella growth control. Taken together, our results provide new insight into how immune responses fight Brucella infection.

Distinct in vivo dendritic cell activation by live versus killed Listeria monocytogenes.

Immunization of mice with live or heat‐killed Listeria monocytogenes (HKLM) efficiently primes pathogen‐specific CD8+ Tu2004cells. T lymphocytes primed by HKLM, however, undergo attenuated proliferation and do not fully differentiate. Thus, only infection with live bacteria induces long‐term, CD8+ Tu2004cell‐mediated protective immunity. In this study we demonstrate that live and heat‐killed bacteria, while both associating with Mac‐3+CD11bhi cells, localize to distinct splenic areas following intravenous inoculation. While HKLM localize to the marginal zone and the splenic red pulp, live L. monocytogenes are carried to the Tu2004cell zone of splenic white pulp. Despite these differences, in vivo depletion of CD11c‐expressing cells prevents priming of naive Tu2004cells by either HKLM or live L. monocytogenes. Analysis of CD11chi dendritic cells (DC) reveals that infection with live L. monocytogenes induces higher levels of CD40, CD80 and CD86 expression than immunization with HKLM. Our results suggest that CD8+ Tu2004cell priming following HKLM immunization or live infection is mediated by DC and that the disparate outcomes of priming can be attributed to suboptimal conditioning of DC in the absence of live, cytosol‐invasive bacteria.

TLR4 and Toll-IL-1 Receptor Domain-Containing Adapter-Inducing IFN-β, but Not MyD88, Regulate Escherichia coli-Induced Dendritic Cell Maturation and Apoptosis In Vivo

Dendritic cells (DC) are short-lived, professional APCs that play a central role in the generation of adaptive immune responses. Induction of efficient immune responses is dependent on how long DCs survive in the host. Therefore, the regulation of DC apoptosis in vivo during infection remains an important question that requires further investigation. The impact of Escherichia coli bacteremia on DCs has never been analyzed. We show here that i.v. or i.p. administration of live or heat-killed E. coli in mice induces splenic DC migration, maturation, and apoptosis. We further characterize which TLR and Toll-IL-1R (TIR)-containing adaptor molecules regulate these processes in vivo. In this model, DC maturation is impaired in TLR2−/−, TLR4−/− and TIR domain-containing adapter-inducing IFN-β (TRIF)−/− mice. In contrast, DC apoptosis is reduced only in TLR4−/− and TRIF−/− mice. As expected, DC apoptosis induced by the TLR4 ligand LPS is also abolished in these mice. Injection of the TLR9 ligand CpG-oligodeoxynucleotide (synthetic bacterial DNA) induces DC migration and maturation, but only modest DC apoptosis when compared with LPS and E. coli. Together, these results suggest that E. coli bacteremia directly impacts on DC maturation and survival in vivo through a TLR4-TRIF-dependent signaling pathway.

TH1/TH2 paradigm extended: macrophage polarization as an unappreciated pathogen-driven escape mechanism?

The classical view of the Th1/Th2 paradigm posits that the pathogen nature, infectious cycle, and persistence represent key parameters controlling the choice of effector mechanisms operating during an immune response. Thus, efficient Th1 responses are triggered by replicating intracellular pathogens, while Th2 responses would control helminth infection and promote tissue repair during the resolution phase of an infectious event. However, this vision does not account for a growing body of data describing how pathogens exploit the polarization of the host immune response to their own benefit. Recently, the study of macrophages has illustrated a novel aspect of this arm race between pathogens and the immune system, and the central role of macrophages in homeostasis, repair and defense of all tissues is now fully appreciated. Like T lymphocytes, macrophages differentiate into distinct effectors including classically (M1) and alternatively (M2) activated macrophages. Interestingly, in addition to represent immune effectors, M1/M2 cells have been shown to represent potential reservoir cells to a wide range of intracellular pathogens. Subversion of macrophage cell metabolism by microbes appears as a recently uncovered immune escape strategy. Upon infection, several microbial agents have been shown to activate host metabolic pathways leading to the production of nutrients necessary to their long-term persistence in host. The purpose of this review is to summarize and discuss the strategies employed by pathogens to manipulate macrophage differentiation, and in particular their basic cell metabolism, to favor their own growth while avoiding immune control.

Lipopolysaccharide-mediatedInterferonRegulatoryFactor Activation Involves TBK1-IKK-dependent Lys 63 -linked Polyubiquitination and Phosphorylation of TANK/I-TRAF *

Type I interferon gene induction relies on IKK-related kinase TBK1 and IKKϵ-mediated phosphorylations of IRF3/7 through the Toll-like receptor-dependent signaling pathways. The scaffold proteins that assemble these kinase complexes are poorly characterized. We show here that TANK/ITRAF is required for the TBK1- and IKKϵ-mediated IRF3/7 phosphorylations through some Toll-like receptor-dependent pathways and is part of a TRAF3-containing complex. Moreover, TANK is dispensable for the early phase of double-stranded RNA-mediated IRF3 phosphorylation. Interestingly, TANK is heavily phosphorylated by TBK1-IKKϵ upon lipopolysaccharide stimulation and is also subject to lipopolysaccharide- and TBK1-IKKϵ-mediated Lys63-linked polyubiquitination, a mechanism that does not require TBK1-IKKϵ kinase activity. Thus, we have identified TANK as a scaffold protein that assembles some but not all IRF3/7-phosphorylating TBK1-IKKϵ complexes and demonstrated that these kinases possess two functions, namely the phosphorylation of both IRF3/7 and TANK as well as the recruitment of an E3 ligase for Lys63-linked polyubiquitination of their scaffold protein, TANK.

DiC14-amidine cationic liposomes stimulate myeloid dendritic cells through Toll-like receptor 4.

DiC14‐amidine cationic liposomes were recently shown to promote Th1 responses when mixed with allergen. To further define the mode of action of diC14‐amidine as potential vaccine adjuvant, we characterized its effects on mouse and human myeloid dendritic cells (DC). First, we observed that, as compared with two other cationic liposomes, only diC14‐amidine liposomes induced the production of IL‐12p40 and TNF‐α by mouse bone marrow‐derived DC. DiC14‐amidine liposomes also activated human DC, as shown by synthesis of IL‐12p40 and TNF‐α, accumulation of IL‐6, IFN‐β and CXCL10 mRNA, and up‐regulation of membrane expression of CD80 and CD86. DC stimulation by diC14‐amidine liposomes was associated with activation of NF‐κB, ERK1/2, JNK and p38 MAP kinases. Finally, we demonstrated in mouse and human cells that diC14‐amidine liposomes use Toll‐like receptor 4 to elicit both MyD88‐dependent and Toll/IL‐1R‐containing adaptor inducing interferon IFN‐β (TRIF)‐dependent responses.

The SH2 domain containing inositol 5-phosphatase SHIP2 associates to the immunoreceptor tyrosine-based inhibition motif of FcγRIIB in B cells under negative signaling

Fc gammaRIIB are single-chain low-affinity receptors for IgG that bear an immunoreceptor tyrosine-based inhibition motif (ITIM) in their intracytoplasmic domain and that negatively regulate immunoreceptor tyrosine-based activation motif (ITAM)-dependent cell activation. In B cells, coaggregation of the B cell receptor (BCR) and Fc gammaRIIB leads to an inhibition of B cell activation. Inhibitory properties of Fc gammaRIIB have been related to the recruitment of SHIP, an SH2 domain-containing inositol 5-phosphatase (referred to as SHIP1), via ITIM phosphorylated Fc gammaRIIB. Here, we demonstrate that the second SH2 domain-containing inositol 5-phosphatase SHIP2 could also bind to the Fc gammaRIIB ITIM. As a model, a Fc gammaRIIB deficient B cell line (IIA1.6), transfected with a cDNA encoding either w.t. Fc gammaRIIB1 or Fc gammaRIIB1 whose ITIM tyrosine was mutated has been used. SHIP2 tyrosine phosphorylation and association to the adaptator protein Shc were only found in transfectants expressing w.t. Fc gammaRIIB1. SHIP2 was also found to bind to a phosphopeptide corresponding to the ITIM sequence of Fc gammaRIIB. There was no binding to the nonphosphorylated peptide. Finally, both SHIP2 and SHIP1 were coprecipitated with Fc gammaRIIB1 upon coaggregation with BCR in IIA1.6 transfectants.