TLR8 on dendritic cells and TLR9 on B cells restrain TLR7-mediated spontaneous autoimmunity in C57BL/6 mice

Systemic lupus erythematosus (SLE) is a complex autoimmune disease with diverse clinical presentations characterized by the presence of autoantibodies to nuclear components. Toll-like receptor (TLR)7, TLR8, and TLR9 sense microbial or endogenous nucleic acids and are implicated in the development of SLE. In mice TLR7-deficiency ameliorates SLE, but TLR8- or TLR9-deficiency exacerbates the disease because of increased TLR7 response. Thus, both TLR8 and TLR9 control TLR7 function, but whether TLR8 and TLR9 act in parallel or in series in the same or different cell types in controlling TLR7-mediated lupus remains unknown. Here, we reveal that double TLR8/9-deficient (TLR8/9^−/−) mice on the C57BL/6 background showed increased abnormalities characteristic of SLE, including splenomegaly, autoantibody production, frequencies of marginal zone and B1 B cells, and renal pathology compared with single TLR8^−/− or TLR9^−/− mice. On the cellular level, TLR8^−/− and TLR8/9^−/− dendritic cells were hyperesponsive to TLR7 ligand R848, but TLR9^−/− cells responded normally. Moreover, B cells from TLR9^−/− and TLR8/9^−/− mice were hyperesponsive to R848, but TLR8^−/− B cells were not. These results reveal that TLR8 and TLR9 have an additive effect on controlling TLR7 function and TLR7-mediated lupus; however, they act on different cell types. TLR8 controls TLR7 function on dendritic cells, and TLR9 restrains TLR7 response on B cells.Systemic lupus erythematosus (SLE) is a complex chronic autoimmune disease that arises spontaneously and is characterized by production of autoantibodies against self-nucleic acids and associated proteins (1). These autoantibodies bind self-nucleic acids released by dying cells and form immune complexes that accumulate in different parts of the body, leading to inflammation and tissue damage. The kidneys, skin, joints, lungs, serous membranes, as well as, the cardiovascular, nervous and musculoskeletal system become targets of inflammation at onset or during the course of the disease (2). The etiology of SLE is unknown, yet genetics, sex, infectious agents, environmental factors, and certain medications may play a role in the initiation of the disease by causing alterations in lymphoid signaling, antigen presentation, apoptosis, and clearance of immune complexes (3, 4).Toll-like receptors (TLRs) detect specific microbial components widely expressed by bacteria, fungi, protozoa, and viruses, and initiate signaling pathways critical for induction of immune responses to infection (5). In contrast to the cell surface TLRs that detect bacterial cell wall components and viral particles, nucleic acid-sensing TLRs are localized mainly within endosomal compartments (6). Human endosomal TLRs consist of TLR3, which senses viral double-stranded RNA (dsRNA) (7), TLR7 and TLR8, which recognize viral single-stranded RNA (8–10), and TLR9, which detects bacterial and viral unmethylated CpG-containing DNA motifs (11). Interestingly, these endosomal TLRs are also able to detect self-nucleic acids (12–14). Although the endosomal localization isolate TLR3, TLR7, TLR8, and TLR9 away from self-nucleic acids in the extracellular space, still self-RNA or -DNA can become a potent trigger of cell activation when transported into TLR-containing endosomes, and such recognition can result in sterile inflammation and autoimmunity, including SLE (4, 15, 16). The connection of the endosomal TLRs with SLE originates mainly from mouse models, where TLR7 signaling seems to play a central role. TLR7 gene duplication is the cause for the development of lupus in mice bearing the Y chromosome-linked autoimmune accelerating (Yaa) locus that harbors 17 genes, including TLR7 (17, 18). In TLR7 transgenic mouse lines, a modest increase in TLR7 expression promotes autoreactive lymphocytes with RNA specificities and myeloid cell proliferation, but a substantial increase in TLR7 expression causes fatal acute inflammatory pathology and profound dendritic cell (DC) dysregulation (17). In addition, studies in several lupus-prone mouse strains have revealed that TLR7-deficiency ameliorates disease, but TLR9-deficiency exacerbates it. Interestingly, this controversy can be explained by the enhanced TLR7 activity in the TLR9-deficient lupus mice (19, 20). Although murine TLR8 does not seem so far to be able to sense a ligand (21, 22), we have shown previously that it plays an important biological role in controlling TLR7-mediated lupus. Indeed, TLR8-deficiency in mice (on the C57BL/6 background that is not prone to lupus) leads to lupus development because of increased TLR7 expression and signaling in DCs (23). Thus, tight control and regulation of TLR7 is pivotal for avoiding SLE and inflammatory pathology in mice. Recent studies in humans have also revealed that increased expression of TLR7 is associated with increased risk for SLE (24–26).Nucleic acid TLRs are expressed in many cell types, including DCs, plasmacytoid DCs (pDCs) and B cells, all of which play a central role in SLE development. TLR7, TLR8, and TLR9 signal through the adaptor molecule myeloid differentiation primary response gene 88 (MyD88), whereas TLR3 signals via the adaptor TRIF (Toll/IL-1 receptor domain-containing adaptor inducing IFN-β) (5). MyD88-deficiency abrogates most attributes of lupus in several lupus-prone mouse strains (19, 27–29). Moreover, deficiency for Unc93B1, a multipass transmembrane protein that controls trafficking of TLRs from the endoplasmic reticulum to endolysosomes and is required for nucleic acid-sensing TLR function (30), also abrogates many clinical parameters of disease in mouse lupus strains, suggesting that endosomal TLRs are critical in this disease (31). Interestingly, TLR9 competes with TLR7 for Unc93B1-dependent trafficking and predominates over TLR7 (32). TLR9 predominance is reversed to TLR7 by a D34A mutation in Unc93B1 and mice that carry this mutation show TLR7-dependent, systemic lethal inflammation (32).Thus, in mice both TLR8 and TLR9 control TLR7-mediated lupus, but it is unknown if these TLRs act in parallel or in series in the same or different cell types and if they have an additive effect or not in controlling TLR7. To address these issues, we generated double TLR8/TLR9-deficient (TLR8/9^−/−) mice and analyzed and compared the lupus phenotype in TLR8^−/−, TLR9^−/−, and TLR8/9^−/− mice. Our data revealed that TLR8/9^−/− mice have increased abnormalities characteristic of SLE and that both TLR8 and TLR9 keep TLR7-mediated lupus under control, but they act in different cell types. On DCs TLR7 function is ruled by TLR8, whereas on B cells TLR7 is mastered by TLR9.     

Development and validation of a multiplex polymerase chain reaction assay for detection of the five families of plasmid-encoded colistin resistance

Plasmid-mediated colistin resistance is increasingly described worldwide in Enterobacteriaceae from animal and human isolates. Diffusion of these resistance traits among carbapenem-resistant enterobacterial isolates is of particular concern as colistin has become the last resort antibiotic for treating human infections with these organisms. Therefore, being able to monitor the presence of these transferable colistin resistance genes (mcr-1 to mcr-5-variants) is crucial. This paper describes the development of a multiplex polymerase chain reaction (PCR) protocol for detection of all currently known transferable colistin resistance genes in Enterobacteriaceae. Five primer pairs were designed to amplify mcr-1, mcr-2, mcr-3, mcr-4 and mcr-5 gene products in a multiplex PCR. This assay was validated retrospectively on colonies of 50 Escherichia coli, 44 Klebsiella pneumoniae and 12 Salmonella enterica isolates of animal and human origin, all well characterized, and validated prospectively on 450 carbapenem-resistant enterobacterial isolates received by the French National Reference Centre. In addition, 82 Aeromonas spp. and 10 Shewanella spp. known to be the progenitors of mcr-3 and mcr-4 alleles, respectively, were screened. Mcr-multiplex PCR assay displayed 100% specificity, sensitivity, negative predictive value and positive predictive value. The assay was able to detect all variants of the different mcr alleles, and was able to detect chromosomally encoded mcr-4-like variants present in two Shewanella bicestrii JAB-1 and Shewanella woodyi S539. In conclusion, a rapid and robust multiplex PCR assay able to detect all known mcr gene families described in Enterobacteriaceae was developed and validated. This type of test is critical for the epidemiological surveillance of plasmid-encoded resistance, especially in carbapenem-resistant bacteria.     

Evaluation of a chromogenic commercial assay using VWF-73 peptide for ADAMTS13 activity measurement

Introduction

Thrombotic thrombocytopenic purpura (TTP) is a thrombotic microangiopathy (TMA), related to a severe functional deficiency of ADAMTS13 activity (< 10% of normal). ADAMTS13 activity is thus crucial to confirm the clinical suspicion of TTP, to distinguish it from other TMAs, and to perform the follow-up of TTP patients.

Material and methods

We compared the performance of the commercial chromogenic assay Technozym® ADAMTS13 Activity ELISA (chromogenic VWF73 substrate, Chr-VWF73, Technoclone, Vienna, Austria), to that of our in-house FRETS-VWF73 used as reference method. A large group of 247 subjects (30 healthy volunteers and 217 patients with miscellaneaous TMAs) was studied.

Results

The lower limit of detection of the Chr-VWF73 was 3%, which is well adapted to the clinically relevant threshold for TTP diagnosis (10%). Our results showed a reasonable agreement between FRETS-VWF73 and Chr-VWF73 assays to distinguish samples with an ADAMTS13 activity < 10% from those with an ADAMTS13 activity > 10%. However, Chr-VWF73 assay provided false negative results in ~ 12% of acute TTP patients. Inversely, the Chr-VWF73 assay globally underestimated ADAMTS13 activity in detectable values ranging from 11 to 100% (with a great variability compared to FRETS-VWF73), which may be a concern for the follow-up of TTP patients in remission.

Conclusion

In-house assays developed and performed by expert laboratories remain the reference methods that should be used without limitation to control values provided by commercial assays when needed. Also, the development of an international reference preparation will be crucial to improve standardization.     

Autophagy is required for CSF-1-induced macrophagic differentiation and acquisition of phagocytic functions

Autophagy is the process by which superfluous or damaged macromolecules or organelles are degraded by the lysosome. Pharmacologic and genetic evidence indicates that autophagy plays pleiotropic functions in cellular homeostasis, development, survival, and differentiation. The differentiation of human blood monocytes into macrophages is a caspase-dependent process when triggered ex vivo by colony stimulating factor-1. We show here, using pharmacologic inhibitors, siRNA approaches, and Atg7-/- mice, that autophagy initiated by ULK1 is required for proper colony stimulating factor-1-driven differentiation of human and murine monocytes. We also unravel a role for autophagy in macrophage acquisition of phagocytic functions. Collectively, these findings highlight an unexpected and essential role of autophagy during monocyte differentiation and acquisition of macrophage functions.     

(-)-Epigallocatechin-3-gallate is a new inhibitor of hepatitis C virus entry

Here, we identify (-)-epigallocatechin-3-gallate (EGCG) as a new inhibitor of hepatitis C virus (HCV) entry. EGCG is a flavonoid present in green tea extract belonging to the subclass of catechins, which has many properties. Particularly, EGCG possesses antiviral activity and impairs cellular lipid metabolism. Because of close links between HCV life cycle and lipid metabolism, we postulated that EGCG may interfere with HCV infection. We demonstrate that a concentration of 50 μM of EGCG inhibits HCV infectivity by more than 90% at an early step of the viral life cycle, most likely the entry step. This inhibition was not observed with other members of the Flaviviridae family tested. The antiviral activity of EGCG on HCV entry was confirmed with pseudoparticles expressing HCV envelope glycoproteins E1 and E2 from six different genotypes. In addition, using binding assays at 4°C, we demonstrate that EGCG prevents attachment of the virus to the cell surface, probably by acting directly on the particle. We also show that EGCG has no effect on viral replication and virion secretion. By inhibiting cell-free virus transmission using agarose or neutralizing antibodies, we show that EGCG inhibits HCV cell-to-cell spread. Finally, by successive inoculation of na?ve cells with supernatant of HCV-infected cells in the presence of EGCG, we observed that EGCG leads to undetectable levels of infection after four passages. CONCLUSION: EGCG is a new, interesting anti-HCV molecule that could be used in combination with other direct-acting antivirals. Furthermore, it is a novel tool to further dissect the mechanisms of HCV entry into the hepatocyte.