Association Between Soluble Notch Ligand Delta-like Ligand 1 and Bleeding Complications in Patients With Dengue Fever Infection.

Bleeding associated with endothelial damage is a key feature of severe dengue fever. In the current study, we investigated whether Notch ligands were associated with bleeding in 115 patients with confirmed dengue infection in Vietnam. Soluble Notch ligands were determined by means of enzyme-linked immunosorbent assay. Seventeen of 115 patients (14.8%) experienced bleeding manifestations. High soluble delta-like ligand 1 (sDLL1) plasma levels was associated with bleeding (median, 15 674 vs 7117 pg/mL; P < .001). Receiver operating characteristic (ROC) curve analysis demonstrated that sDLL1 had the best test performance (area under the ROC curve, 0.852), with 88% sensitivity and 84% specificity. The combination with alanine aminotransferase and aspartate aminotransferase slightly increased sDLL1 performance. sDLL1 may be useful to guide clinical management of patients with patients in endemic settings.

Host factors facilitating SARS-CoV-2 virus infection and replication in the lungs.

SARS-CoV-2 is the virus causing the major pandemic facing the world today. Although, SARS-CoV-2 primarily causes lung infection, a variety of symptoms have proven a systemic impact on the body. SARS-CoV-2 has spread in the community quickly infecting humans from all age, ethnicities and gender. However, fatal outcomes have been linked to specific host factors and co-morbidities such as age, hypertension, immuno-deficiencies, chronic lung diseases or metabolic disorders. A major shift in the microbiome of patients suffering of the coronavirus disease 2019 (COVID-19) have also been observed and is linked to a worst outcome of the disease. As many co-morbidities are already known to be associated with a dysbiosis of the microbiome such as hypertension, diabetes and metabolic disorders. Host factors and microbiome changes are believed to be involved as a network in the acquisition of the infection and the development of the diseases. We will review in detail in this manuscript, the immune response toward SARS-CoV-2 infection as well as the host factors involved in the facilitation and worsening of the infection. We will also address the impact of COVID-19 on the host's microbiome and secondary infection which also worsen the disease.

Pitfalls in genotypic antimicrobial susceptibility testing caused by low expression of blaKPC in Escherichia coli.

BACKGROUND: There is a growing interest in the rapid genotypic identification of antimicrobial resistance (AMR). In routine diagnostics, we detected multiple KPC-positive Escherichia coli (KPC-Ec) with discordant phenotypic meropenem susceptibility from a single patient's blood cultures, which prompted a more thorough investigation. OBJECTIVES: We investigated the potential clinical relevance of, and the mechanism behind, discordant phenotypic and genotypic meropenem susceptibility in KPC-Ec. METHODS: WGS was used to perform a comparative analysis of the isolates' genetic characteristics and their blaKPC-2 locus. Expression of blaKPC-2 was determined by quantitative PCR and the potency of meropenem hydrolysis was determined using a semi-quantitative carbapenem inactivation method. An in vivo infection assay using Galleria mellonella was performed to assess the potential clinical relevance of KPC expression in E. coli. RESULTS: Despite the presence of blaKPC-2, three of five isolates were susceptible to meropenem (MICVITEK2 ≤ 0.25 mg/L), while two isolates were resistant (MICVITEK2 ≥ 16 mg/L). The isolates with high MICs had significantly higher blaKPC-2 expression, which corresponds to phenotypic meropenem inactivation. The genetic environment of blaKPC-2, which may impact KPC production, was identical in all isolates. In vivo infection assay with G. mellonella suggested that meropenem was effective in reducing mortality following infection with low-expressing KPC-Ec. CONCLUSIONS: Our findings clearly highlight a limitation of genotypic AMR prediction for blaKPC. For the time being, genotypic AMR prediction requires additional analysis for accurate antibiotic therapy decision-making.

Streptococcal Pyrogenic Exotoxin A-Stimulated Monocytes Mediate Regulatory T-Cell Accumulation through PD-L1 and Kynurenine.

Bacterial superantigens (SAgs) are exotoxins that promote a fulminant activation of the immune system. The subsequent intense release of inflammatory cytokines often results in hypotension, shock, and organ failure with high mortality rates. In the current paradigm, the direct and simultaneous binding of SAgs with T-cell receptor (TCR)-bearing Vß regions and conserved structures on major histocompatibility complex class II (MHC class II) on antigen-presenting cells (APCs) induces the activation of both cell types. However, by crosslinking MHC class II molecules, APCs can be activated by SAgs independently of T lymphocytes. Recently, we showed that streptococcal pyrogenic exotoxin A (SPEA) of Streptococcus pyogenes stimulates an immunogenic APC phenotype with upregulated costimulatory molecules and inflammatory cytokines. Additionally, we revealed that SPEA triggers immunosuppressive programs in monocytes that facilitate the accumulation of regulatory T cells (Tregs) in in vitro monocyte/CD4+ T-cell cocultures. Immunosuppressive factors include anti-inflammatory interleukin 10 (IL-10), co-inhibitory surface molecule programmed cell death 1 ligand 1 (PD-L1), and the inhibitory indoleamine 2,3-dioxygenase (IDO)/kynurenine effector system. In the present study, we investigated the underlying mechanism of SPEA-stimulated monocyte-mediated accumulation of Tregs. Blood-derived monocytes from healthy donors were stimulated with SPEA for 48 h (SPEA-monocytes). For the evaluation of SPEA-monocyte-mediated modulation of CD4+ T lymphocytes, SPEA was removed from the culture through extensive washing of cells before adding allogeneic CD3/CD28-activated T cells. Results: In coculture with allogeneic CD4+ T cells, SPEA-monocytes mediate apoptosis of CD4+Foxp3- lymphocytes and accumulation of CD4+Foxp3+ Tregs. PD-L1 and kynurenine are critically involved in the mediated cell death because blocking both factors diminished apoptosis and decreased the proportion of the CD25+/Foxp3+ Treg subpopulation significantly. Upregulation of PD-L1 and kynurenine as well as SPEA-monocyte-mediated effects on T cells depend on inflammatory IL-1ß. Our study shows that monocytes activated by SPEA mediate apoptosis of CD4+Foxp3- T effector cells through PD-L1 and kynurenine. CD4+Foxp3+ T cells are resistant to apoptosis and accumulate in SPEA-monocyte/CD4+ T-cell coculture.

Analysis of the interplay between all-trans retinoic acid and histone deacetylase inhibitors in leukemic cells.

The treatment of acute promyelocytic leukemia (APL) with all-trans retinoic acid (ATRA) induces granulocytic differentiation. This process renders APL cells resistant to cytotoxic chemotherapies. Epigenetic regulators of the histone deacetylases (HDACs) family, which comprise four classes (I-IV), critically control the development and progression of APL. We set out to clarify the parameters that determine the interaction between ATRA and histone deacetylase inhibitors (HDACi). Our assays included drugs against class I HDACs (MS-275, VPA, and FK228), pan-HDACi (LBH589, SAHA), and the novel HDAC6-selective compound Marbostat-100. We demonstrate that ATRA protects APL cells from cytotoxic effects of SAHA, MS-275, and Marbostat-100. However, LBH589 and FK228, which have a superior substrate-inhibitor dissociation constant (Ki) for the class I deacetylases HDAC1, 2, 3, are resistant against ATRA-dependent cytoprotective effects. We further show that HDACi evoke DNA damage, measured as induction of phosphorylated histone H2AX and by the comet assay. The ability of ATRA to protect APL cells from the induction of p-H2AX by HDACi is a readout for the cytoprotective effects of ATRA. Moreover, ATRA increases the fraction of cells in the G1 phase, together with an accumulation of the cyclin-dependent kinase inhibitor p21 and a reduced expression of thymidylate synthase (TdS). In contrast, the ATRA-dependent activation of the transcription factors STAT1, NF-κB, and C/EBP hardly influences the responses of APL cells to HDACi. We conclude that the affinity of HDACi for class I HDACs determines whether such drugs can kill naïve and maturated APL cells.

Corrigendum: Inflammatory response against Staphylococcus aureus via intracellular sensing of nucleic acids in keratinocytes.

[This corrects the article DOI: 10.3389/fimmu.2022.828626.].

Regulation of Toll-like receptor 4-mediated immune responses through Pasteurella multocida toxin-induced G protein signalling.

BACKGROUND: Lipopolysaccharide (LPS)-triggered Toll-like receptor (TLR) 4-signalling belongs to the key innate defence mechanisms upon infection with Gram-negative bacteria and triggers the subsequent activation of adaptive immunity. There is an active crosstalk between TLR4-mediated and other signalling cascades to secure an effective immune response, but also to prevent excessive inflammation. Many pathogens induce signalling cascades via secreted factors that interfere with TLR signalling to modify and presumably escape the host response. In this context heterotrimeric G proteins and their coupled receptors have been recognized as major cellular targets. Toxigenic strains of Gram-negative Pasteurella multocida produce a toxin (PMT) that constitutively activates the heterotrimeric G proteins Gαq, Gα13 and Gαi independently of G protein-coupled receptors through deamidation. PMT is known to induce signalling events involved in cell proliferation, cell survival and cytoskeleton rearrangement. RESULTS: Here we show that the activation of heterotrimeric G proteins through PMT suppresses LPS-stimulated IL-12p40 production and eventually impairs the T cell-activating ability of LPS-treated monocytes. This inhibition of TLR4-induced IL-12p40 expression is mediated by Gαi-triggered signalling as well as by Gßγ-dependent activation of PI3kinase and JNK.Taken together we propose the following model: LPS stimulates TLR4-mediated activation of the NFĸB-pathway and thereby the production of TNF-α, IL-6 and IL-12p40. PMT inhibits the production of IL-12p40 by Gαi-mediated inhibition of adenylate cyclase and cAMP accumulation and by Gßγ-mediated activation of PI3kinase and JNK activation. CONCLUSIONS: On the basis of the experiments with PMT this study gives an example of a pathogen-induced interaction between G protein-mediated and TLR4-triggered signalling and illustrates how a bacterial toxin is able to interfere with the host's immune response.

Inflammatory Response Against Staphylococcus aureus via Intracellular Sensing of Nucleic Acids in Keratinocytes.

Staphylococcus aureus is one of the clinically most relevant pathogens causing infections. Humans are often exposed to S. aureus. In approximately one-third of the healthy population it can be found on the skin either for long or short periods as colonizing "commensals", without inducing infections or an inflammatory immune response. While tolerating S. aureus seems to be limited to certain individuals and time periods in most cases, Staphylococcus epidermidis is tolerated permanently on the skin of almost all individuals without activating overwhelming skin inflammation. To investigate this, we co-cultured a keratinocyte cell line (HaCaT) with viable S. aureus or S. epidermidis to study the differences in the immune activation. S. aureus activated keratinocytes depicted by a profound IL-6 and IL-8 response, whereas S. epidermidis did not. Our data indicate that internalization of S. aureus and the subsequent intracellular sensing of bacterial nucleic acid may be essential for initiating inflammatory response in keratinocytes. Internalized dsRNA activates IL-6 and IL-8 release, but not TNF-α or IFNs by human keratinocytes. This is a non-specific effect of dsRNA, which can be induced using Poly(I:C), as well as RNA from S. aureus and S. epidermidis. However, only viable S. aureus were able to induce this response as these bacteria and not S. epidermidis were actively internalized by HaCaT. The stimulatory effect of S. aureus seems to be independent of the TLR3, -7 and -8 pathways.

Cefiderocol Protects against Cytokine- and Endotoxin-Induced Disruption of Vascular Endothelial Cell Integrity in an In Vitro Experimental Model.

The severe course of bloodstream infections with Gram-negative bacilli can lead to organ dysfunctions and compromise the integrity of the vascular barrier, which are the hallmarks of sepsis. This study aimed to investigate the potential effect of cefiderocol on the barrier function of vascular endothelial cells (vECs) in an in vitro experimental set-up. Human umbilical vein cells (HUVECs), co-cultured with erythrocyte-depleted whole blood for up to 48 h, were activated with tumor necrosis factor-alpha (TNF-α) or lipopolysaccharide (LPS) to induce endothelial damage in the absence or presence of cefiderocol (concentrations of 10, 40 and 70 mg/L). The endothelial integrity was quantified using transendothelial electrical resistance (TEER) measurement, performed at 0, 3, 24 and 48 h after stimulation. Stimulation with TNF-α and LPS increased the endothelial permeability assessed by TEER at 24 and 48 h of co-culture. Furthermore, cefiderocol reduces interleukin-6 (IL-6), interleukin-1ß (IL-1ß) and TNF-α release in peripheral blood mononuclear cells (PBMCs) following LPS stimulation in a dose-dependent manner. Collectively, the data suggest that cefiderocol may have an influence on the cellular immune response and might support the maintenance of vEC integrity during inflammation associated with infection with Gram-negative bacteria, which warrants further investigations.

Pasteurella multocida toxin-stimulated osteoclast differentiation is B cell dependent.

Pasteurella multocida is a Gram-negative bacillus that infects a number of wild and domestic animals, causing respiratory diseases. Toxigenic Pasteurella multocida strains produce a protein toxin (PMT) that leads to atrophic rhinitis in swine due to enhanced osteoclastogenesis and the inhibition of osteoblast function. We show that PMT-induced osteoclastogenesis is promoted by an as-yet-uncharacterized B-cell population. The toxin, however, is not acting at the level of hematopoietic stem cells, since purified CD117(+) cells from murine hematopoietic progenitor cells cultivated with PMT did not mature into osteoclasts. The early macrophages contained within this cell population (CD117(+)/CD11b(+)) did not further differentiate into osteoclasts but survived and were able to phagocytose. Within the CD117(-) population, however, we detected PMT-induced generation of a B220(+)/CD19(+) and B220(+)/IgM(+) B-cell population that was able to take up fluorescently labeled PMT. Using purified B-cell and macrophage populations, we show that these B cells are needed to efficiently generate osteoclasts from macrophages. Cells of the immune system are thought to affect osteoclast formation and function by secreting cytokines and growth factors. We show here that PMT-stimulated B cells produce elevated levels of the osteoclastogenic factors interleukin-1ß (IL-1ß), IL-6, tumor necrosis factor alpha, and receptor activator of nuclear factor receptor ligand (RANKL) compared to B cells generated through incubation with IL-7. These results suggest that the osteoclastic properties characteristic for PMT may result from a cross talk between bone cells and lymphoid cells and that B cells might be an important target of Pasteurella multocida.