RadA, a Key Gene of the Circadian Rhythm of Escherichia coli.

Circadian rhythms are present in almost all living organisms, and their activity relies on molecular clocks. In prokaryotes, a functional molecular clock has been defined only in cyanobacteria. Here, we investigated the presence of circadian rhythms in non-cyanobacterial prokaryotes. The bioinformatic approach was used to identify a homologue of KaiC (circadian gene in cyanobacteria) in Escherichia coli. Then, strains of E. coli (wild type and mutants) were grown on blood agar, and sampling was made every 3 h for 24 h at constant conditions. Gene expression was determined by qRT-PCR, and the rhythmicity was analyzed using the Cosinor model. We identified RadA as a KaiC homologue in E. coli. Expression of radA showed a circadian rhythm persisting at least 3 days, with a peak in the morning. The circadian expression of other E. coli genes was also observed. Gene circadian oscillations were lost in radA mutants of E. coli. This study provides evidence of molecular clock gene expression in E. coli with a circadian rhythm. Such a finding paves the way for new perspectives in antibacterial treatment.

Monocytes and Macrophages, Targets of Severe Acute Respiratory Syndrome Coronavirus 2: The Clue for Coronavirus Disease 2019 Immunoparalysis.

BACKGROUND: Coronavirus disease 2019 (COVID-19) clinical expression is pleiomorphic, severity is related to age and comorbidities such as diabetes and hypertension, and pathophysiology involves aberrant immune activation and lymphopenia. We wondered if the myeloid compartment was affected during COVID-19 and if monocytes and macrophages could be infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). METHODS: Monocytes and monocyte-derived macrophages (MDMs) from COVID-19 patients and controls were infected with SARS-CoV-2 and extensively investigated with immunofluorescence, viral RNA extraction and quantification, and total RNA extraction followed by reverse-transcription quantitative polymerase chain reaction using specific primers, supernatant cytokines (interleukins 6, 10, and 1ß; interferon-ß; transforming growth factor-ß1, and tumor necrosis factor-α), and flow cytometry. The effect of M1- vs M2-type or no polarization prior to infection was assessed. RESULTS: SARS-CoV-2 efficiently infected monocytes and MDMs, but their infection is abortive. Infection was associated with immunoregulatory cytokines secretion and the induction of a macrophagic specific transcriptional program characterized by the upregulation of M2-type molecules. In vitro polarization did not account for permissivity to SARS-CoV-2, since M1- and M2-type MDMs were similarly infected. In COVID-19 patients, monocytes exhibited lower counts affecting all subsets, decreased expression of HLA-DR, and increased expression of CD163, irrespective of severity. CONCLUSIONS: SARS-CoV-2 drives monocytes and macrophages to induce host immunoparalysis for the benefit of COVID-19 progression.SARS-CoV-2 infection of macrophages induces a specific M2 transcriptional program. In Covid-19 patients, monocyte subsets were decreased associated with up-expression of the immunoregulatory molecule CD163 suggesting that SARS-CoV-2 drives immune system for the benefit of Covid-19 disease progression.

Phenotypic diversity of Tropheryma whipplei clinical isolates.

Tropheryma whipplei is a bacterial pathogen responsible for a wide range of infections in humans, covering asymptomatic carriage, acute infections, chronic isolated infections and classic Whipple's disease. Although the bacterium is commonly found in the environment, it very rarely causes disease. Genetic comparison of clinical isolates has revealed that main variations were found in region encoding T. whipplei surface glycoproteins called WiSP. However, no association has been made between the genetic diversity and the clinical manifestations of the infection. In this study we evaluated the phenotypic diversity of 26 clinical isolates from different origins and taken from patient with different infection outcomes. MRC5 and macrophages cells were infected, and bacterial uptake, survival and the pro-and anti-inflammatory potential of the different clinical isolates was assessed. No significant difference of phagocytosis was found between the different isolates; however, we found that bacterial replication was increased for bacteria expressing high molecular weight WiSP. In addition, we found that the expression of the genes coding for IL-1ß and TGF-ß was significantly higher when MRC5 cells were stimulated with isolates from chronic infections compared to isolates from localized infections while no significant differences were observed in macrophages. Overall, our study revealed that, as previously observed at the genetic level, phenotypic diversity of T. whipplei isolates is associated with the expression of different WiSP, which may result in subtle differences in host responses. Other host factors or genetic predisposition may explain the range of clinical manifestations of T. whipplei infections.

Tropheryma whipplei Increases Expression of Human Leukocyte Antigen-G on Monocytes to Reduce Tumor Necrosis Factor and Promote Bacterial Replication.

BACKGROUND & AIMS: Infection with Tropheryma whipplei has a range of effects-some patients can be chronic carriers without developing any symptoms, whereas others can develop systemic Whipple disease, characterized by a lack a protective inflammatory immune response. Alterations in HLA-G function have been associated with several diseases. We investigated the role of HLA-G during T whipplei infection. METHODS: Sera, total RNA, and genomic DNA were collected from peripheral blood from 22 patients with classic Whipple's disease, 19 patients with localized T whipplei infections, and 21 asymptomatic carriers. Levels of soluble HLA-G in sera were measured by enzyme-linked immuosorbent assay, and expressions of HLA-G and its isoforms were monitored by real-time polymerase chain reaction. HLA-G alleles were identified and compared with a population of voluntary bone marrow donors. Additionally, monocytes from healthy subjects were stimulated with T whipplei, and HLA-G expression was monitored by real-time polymerase chain reaction and flow cytometry. Bacterial replication was assessed by polymerase chain reaction in the presence of HLA-G or inhibitor of tumor necrosis factor (TNF) (etanercept). RESULTS: HLA-G mRNAs and levels of soluble HLA-G were significantly increased in sera from patients with chronic T whipplei infection compared with sera from asymptomatic carriers and control individuals. No specific HLA-G haplotypes were associated with disease or T whipplei infection. However, T whipplei infection of monocytes induced expression of HLA-G, which was associated with reduced secretion of TNF compared with noninfected monocytes. A neutralizing antibody against HLA-G increased TNF secretion by monocytes in response to T whipplei, and a TNF inhibitor promoted bacteria replication. CONCLUSIONS: Levels of HLA-G are increased in sera from patients with T whipplei tissue infections, associated with reduced production of TNF by monocytes. This might promote bacteria colonization in patients.

The transcriptional repressor Gfi1 prevents lupus autoimmunity by restraining TLR7 signaling.

The transcriptional repressor growth factor independence 1 (Gfi1) is important in myeloid and lymphoid differentiation. In the current study we evaluated the involvement of Gfi1 in systemic lupus erythematosus (SLE). We found that Genista mice, which carry a hypomorphic mutation in the gfi1 gene or Gfi1-deficient (Gfi1-/- ) mice develop signs of spontaneous lupus autoimmunity, including increased serum levels of IgM and IgG2a, autoantibodies against RNA and DNA, glomerular immunodeposits and increased frequencies of plasmablasts, germinal center (GC) B cells and age-associated B cells (ABCs). On the contrary, Genista mice deprived of TLR7 did not show any of these phenotypes, suggesting that the observed lupus autoimmunity in Genista mice is TLR7-dependent. Moreover, Genista mice showed an increased activation of dendritic cells (DCs), B and T cells that was dependent on TLR7 for DCs and B cells, but not for T cells. Upon TLR7 or TLR4 stimulation Genista DCs produced increased amounts of TNF, IL-6 and IFN-ß and showed increased NF-κB phosphorylation and IRF7 nuclear translocation, suggesting that Gfi1 controls the NF-κB and type I IFN signaling pathway downstream of TLRs. Our data reveal that Gfi1 plays a critical role in the prevention of spontaneous lupus autoimmunity by negatively regulating TLR7 signaling.

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.

Vimentin: from a cytoskeletal protein to a critical modulator of immune response and a target for infection.

Vimentin is an intermediate filament protein that plays a role in cell processes, including cell migration, cell shape and plasticity, or organelle anchorage. However, studies from over the last quarter-century revealed that vimentin can be expressed at the cell surface and even secreted and that its implications in cell physiology largely exceed structural and cytoskeletal functions. Consequently, vimentin contributes to several pathophysiological conditions such as cancer, autoimmune and inflammatory diseases, or infection. In this review, we aimed at covering these various roles and highlighting vimentin implications in the immune response. We also provide an overview of how some microbes including bacteria and viruses have acquired the ability to circumvent vimentin functions in order to interfere with host responses and promote their uptake, persistence, and egress from host cells. Lastly, we discuss the therapeutic approaches associated with vimentin targeting, leading to several beneficial effects such as preventing infection, limiting inflammatory responses, or the progression of cancerous events.

Type I interferon induction is detrimental during infection with the Whipple's disease bacterium, Tropheryma whipplei.

Macrophages are the first line of defense against pathogens. Upon infection macrophages usually produce high levels of proinflammatory mediators. However, macrophages can undergo an alternate polarization leading to a permissive state. In assessing global macrophage responses to the bacterial agent of Whipple's disease, Tropheryma whipplei, we found that T. whipplei induced M2 macrophage polarization which was compatible with bacterial replication. Surprisingly, this M2 polarization of infected macrophages was associated with apoptosis induction and a functional type I interferon (IFN) response, through IRF3 activation and STAT1 phosphorylation. Using macrophages from mice deficient for the type I IFN receptor, we found that this type I IFN response was required for T. whipplei-induced macrophage apoptosis in a JNK-dependent manner and was associated with the intracellular replication of T. whipplei independently of JNK. This study underscores the role of macrophage polarization in host responses and highlights the detrimental role of type I IFN during T. whipplei infection.

[Toll-like receptor 8: the awkward TLR]. / Le récepteur Toll-like 8 - Un TLR pas comme les autres.

Toll-like receptors (TLR) sense a variety of microbial products and play an important role in the mounting of innate and adaptive immune responses. TLR1 to TLR9 are common in mice and humans and recognize similar ligands in both species, with the exception of TLR8. Human TLR7 and TLR8 and mouse TLR7 detect viral single-stranded RNA and imidazoquinoline compounds, while mouse TLR8 not. Based on this discrepancy, for long time it was believed that mouse TLR8 is not functional and as a consequence the contribution of TLR8 to innate immunity remained poorly understood. Our recent studies revealed an important role for TLR8 in the regulation of TLR7-mediated autoimmunity in the mouse. This review illustrates our current understanding regarding the function of TLR8 and its potential for future clinical use for the treatment and/or prevention of various pathological conditions.

An experimental mouse model to establish Tropheryma whipplei as a diarrheal agent.

Tropheryma whipplei has long been considered as a rare bacterium causing a rare disease, Whipple's disease. However, recent advances now suggest that T. whipplei is a ubiquitous environmental bacterium that may cause gastroenteritis, commonly associated with viral pathogens. We developed an animal model to support this hypothesis. We found that orally given T. whipplei induced diarrhea in mice, without spreading into the intestines. Aggravating factors, such as damage to the intestinal mucosa, favored bacterial spreading. Indeed, bacterial presence was prolonged in stools of dextran sulfate-treated mice, and bacteria were detected in the colon. This resulted in an immune response, with T. whipplei-specific serum IgM and IgG and fecal IgA, as measured by newly introduced immuno-polymerase chain reaction technique. Our results confirm that T. whipplei is an agent causing gastroenteritis and suggest that existing mucosal damage may favor bacterial invasion of tissues.

Vimentin is an important ACE2 co-receptor for SARS-CoV-2 in epithelial cells.

Vimentin is a type III intermediate filament protein, widely expressed in mesenchymal cells. Mainly located in the cytoplasm, vimentin can also appear at extracellular locations, where it may interact with bacterial or viral pathogens. In this study, we aimed at investigating the implication of vimentin in SARS-CoV-2 viral entry and the consequences on viral replication and cellular response. We showed that upon infection, vimentin was upregulated at the cell surface, where it interacts with ACE2 for SARS-CoV-2 entry. We demonstrated a direct interaction between SARS-CoV-2 spike protein, ACE2, and vimentin in epithelial cells. Inhibition of cell-surface vimentin availability resulted in reduced viral entry and cytopathogenic effects. Finally, we showed that the expression of inflammatory cytokines and chemokines was modulated by vimentin-SARS-CoV-2 interaction. In conclusion, our data suggest that cell-surface vimentin acts as a co-receptor for SARS-CoV-2.

Whipple's disease and Tropheryma whipplei infections: from bench to bedside.

Whipple's disease is a chronic and systemic disease caused by the Gram-positive bacterium Tropheryma whipplei that primarily affects the gastrointestinal tract. Data from the last two decades have substantially increased our knowledge of the spectrum and our understanding of T whipplei infections. Although T whipplei seems ubiquitously present in the environment, Whipple's disease itself is very rare. Remarkably, primary infections can be symptomatic, but most cases result in bacterial clearance and seroconversion. However, some individuals are unable to clear the bacterium leading to persistence and asymptomatic carriage. In very rare cases, which might be associated with a subtle immune defect, T whipplei replication is uncontrolled and manifests as classical Whipple's disease or T whipplei localised infections. In this review, we provide a comprehensive outline of T whipplei infection, including the epidemiology, clinical manifestations, diagnosis, and treatment. We also provide an up-to-date overview of our understanding of the host immune response and pathophysiology and discuss future research avenues to resolve the lacking pieces of the puzzle of T whipplei infections.

A Massive Number of Extracellular Tropheryma whipplei in Infective Endocarditis: A Case Report and Literature Review.

Whipple's disease (WD) is a chronic multisystemic infection caused by Tropheryma whipplei. If this bacterium presents an intracellular localization, associated with rare diseases and without pathognomonic signs, it is often subject to a misunderstanding of its physiopathology, often a misdiagnosis or simply an oversight. Here, we report the case of a patient treated for presumed rheumatoid arthritis. Recently, this patient presented to the hospital with infectious endocarditis. After surgery and histological analysis, we discovered the presence of T. whipplei. Electron microscopy allowed us to discover an atypical bacterial organization with a very large number of bacteria present in the extracellular medium in vegetation and valvular tissue. This atypical presentation we report here might be explained by the anti-inflammatory treatment administrated for our patient's initial diagnosis of rheumatoid arthritis.

Tumor Necrosis Factor Inhibitors Exacerbate Whipple's Disease by Reprogramming Macrophage and Inducing Apoptosis.

Tropheryma whipplei is the agent of Whipple's disease, a rare systemic disease characterized by macrophage infiltration of the intestinal mucosa. The disease first manifests as arthralgia and/or arthropathy that usually precede the diagnosis by years, and which may push clinicians to prescribe Tumor necrosis factor inhibitors (TNFI) to treat unexplained arthralgia. However, such therapies have been associated with exacerbation of subclinical undiagnosed Whipple's disease. The objective of this study was to delineate the biological basis of disease exacerbation. We found that etanercept, adalimumab or certolizumab treatment of monocyte-derived macrophages from healthy subjects significantly increased bacterial replication in vitro without affecting uptake. Interestingly, this effect was associated with macrophage repolarization and increased rate of apoptosis. Further analysis revealed that in patients for whom Whipple's disease diagnosis was made while under TNFI therapy, apoptosis was increased in duodenal tissue specimens as compared with control Whipple's disease patients who never received TNFI prior diagnosis. In addition, IFN-γ expression was increased in duodenal biopsy specimen and circulating levels of IFN-γ were higher in patients for whom Whipple's disease diagnosis was made while under TNFI therapy. Taken together, our findings establish that TNFI aggravate/exacerbate latent or subclinical undiagnosed Whipple's disease by promoting a strong inflammatory response and apoptosis and confirm that patients may be screened for T. whipplei prior to introduction of TNFI therapy.

Impact of Sex Hormones on Macrophage Responses to Coxiella burnetii.

Introduction: Q fever, a zoonosis caused by Coxiella burnetii, affects more males than females despite a similar level of exposure. A protective role of estradiol has been reported in mice, suggesting that sex hormones are involved in C. burnetii infection. We wondered whether the responses of monocytes and monocyte-derived macrophages (MDMs) to C. burnetii are influenced by sex hormones. Materials and Methods: The bacterial intracellular fate in monocytes was studied using quantitative PCR, and monocyte cytokine production in response to C. burnetii was assessed using qRT-PCR and immunoassays. Before infection, MDMs from males and females were incubated with testosterone and estradiol, respectively. Results: Bacterial uptake and persistence were similar in monocytes from males and females but were slightly increased in male MDMs. The expression of inflammatory genes, including those encoding TNF and CXCL10, was higher in MDMs from females than in MDMs from males infected by C. burnetii. Adding testosterone to male MDMs amplified their immunoregulatory properties, including increased expression of IL10 and TGFB genes and TGF-ß production in response to C. burnetii. In contrast, adding estradiol to MDMs from females had no effect on their inflammatory profile. Conclusion: The stronger inflammatory profile of macrophages from females may have a protective role, likely under estrogen control, while testosterone may affect disease progression by promoting an anti-inflammatory response. This finding may have consequences for personalized management of patients with Q fever.

Human galectin-1 and galectin-3 promote Tropheryma whipplei infection.

Tropheryma whipplei, is an actinobacterium that causes different infections in humans, including Whipple's disease. The bacterium infects and replicates in macrophages, leading to a Th2-biased immune response. Previous studies have shown that T. whipplei harbors complex surface glycoproteins with evidence of sialylation. However, the exact contribution of these glycoproteins for infection and survival remains obscure. To address this, we characterized the bacterial glycoprofile and evaluated the involvement of human ß-galactoside-binding lectins, Galectin-1 (Gal-1) and Galectin-3 (Gal-3) which are highly expressed by macrophages as receptors for bacterial glycans. Tropheryma whipplei glycoproteins harbor different sugars including glucose, mannose, fucose, ß-galactose and sialic acid. Mass spectrometry identification revealed that these glycoproteins were membrane- and virulence-associated glycoproteins. Most of these glycoproteins are highly sialylated and N-glycosylated while some of them are rich in poly-N-acetyllactosamine (Poly-LAcNAc) and bind Gal-1 and Gal-3. In vitro, T. whipplei modulates the expression and cellular distribution of Gal-1 and Gal-3. Although both galectins promote T. whipplei infection by enhancing bacterial cell entry, only Gal-3 is required for optimal bacterial uptake. Finally, we found that serum levels of Gal-1 and Gal-3 were altered in patients with T. whipplei infections as compared to healthy individuals, suggesting that galectins are also involved in vivo. Among T. whipplei membrane-associated proteins, poly-LacNAc rich-glycoproteins promote infection through interaction with galectins. T. whipplei modulates the expression of Gal-1 and Gal-3 both in vitro and in vivo. Drugs interfering with galectin-glycan interactions may provide new avenues for the treatment and diagnosis of T. whipplei infections.

Macrophage polarization in bacterial infections.

Converging studies have shown that M1 and M2 macrophages are functionally polarized in response to microorganisms and host mediators. Gene expression profiling of macrophages reveals that various Gram-negative and Gram-positive bacteria induce the transcriptional activity of a "common host response," which includes genes belonging to the M1 program. However, excessive or prolonged M1 polarization can lead to tissue injury and contribute to pathogenesis. The so-called M2 macrophages play a critical role in the resolution of inflammation by producing anti-inflammatory mediators. These M2 cells cover a continuum of cells with different phenotypic and functional properties. In addition, some bacterial pathogens induce specific M2 programs in macrophages. In this review, we discuss the relevance of macrophage polarization in three domains of infectious diseases: resistance to infection, infectious pathogenesis, and chronic evolution of infectious diseases.

Utilization of Galectins by Pathogens for Infection.

Galectins are glycan-binding proteins which are expressed by many different cell types and secreted extracellularly. These molecules are well-known regulators of immune responses and involved in a broad range of cellular and pathophysiological functions. During infections, host galectins can either avoid or facilitate infections by interacting with host cells- and/or pathogen-derived glycoconjugates and less commonly, with proteins. Some pathogens also express self-produced galectins to interfere with host immune responses. This review summarizes pathogens which take advantage of host- or pathogen-produced galectins to establish the infection.

Immune Modulation as a Therapeutic Option During the SARS-CoV-2 Outbreak: The Case for Antimalarial Aminoquinolines.

The rapid spread, severity, and lack of specific treatment for COVID-19 resulted in hasty drug repurposing. Conceptually, trials of antivirals were well-accepted, but twentieth century antimalarials sparked an impassioned global debate. Notwithstanding, antiviral and immunomodulatory effects of aminoquinolines have been investigated in vitro, in vivo and in clinical trials for more than 30 years. We review the mechanisms of action of (hydroxy)chloroquine on immune cells and networks and discuss promises and pitfalls in the fight against SARS-CoV-2, the agent of the COVID-19 outbreak.

CNF1-induced ubiquitylation and proteasome destruction of activated RhoA is impaired in Smurf1-/- cells.

Ubiquitylation of RhoA has emerged as an important aspect of both the virulence of Escherichia coli producing cytotoxic necrotizing factor (CNF) 1 toxin and the establishment of the polarity of eukaryotic cells. Owing to the molecular activity of CNF1, we have investigated the relationship between permanent activation of RhoA catalyzed by CNF1 and subsequent ubiquitylation of RhoA by Smurf1. Using Smurf1-deficient cells and by RNA interference (RNAi)-mediated Smurf1 knockdown, we demonstrate that Smurf1 is a rate-limiting and specific factor of the ubiquitin-mediated proteasomal degradation of activated RhoA. We further show that the cancer cell lines HEp-2, human embryonic kidney 293 and Vero are specifically deficient in ubiquitylation of either activated Rac, Cdc42, or Rho, respectively. In contrast, CNF1 produced the cellular depletion of all three isoforms of Rho proteins in the primary human cell types we have tested. We demonstrate that ectopic expression of Smurf1 in Vero cells, deficient for RhoA ubiquitylation, restores ubiquitylation of the activated forms of RhoA. We conclude here that Smurf1 ubiquitylates activated RhoA and that, in contrast to human primary cell types, some cancer cell lines have a lower ubiquitylation capacity of specific Rho proteins. Thus, both CNF1 and transforming growth factor-beta trigger activated RhoA ubiquitylation through Smurf1 ubiquitin-ligase.