Simultaneous Targeting of IL-1-Signaling and IL-6-Trans-Signaling Preserves Human Pulmonary Endothelial Barrier Function During a Cytokine Storm-Brief Report.

BACKGROUND: Systemic inflammatory diseases, such as sepsis and severe COVID-19, provoke acute respiratory distress syndrome in which the pathological hyperpermeability of the microvasculature, induced by uncontrolled inflammatory stimulation, causes pulmonary edema. Identifying the inflammatory mediators that induce human lung microvascular endothelial cell barrier dysfunction is essential to find the best anti-inflammatory treatments for critically ill acute respiratory distress syndrome patients. METHODS: We have compared the responses of primary human lung microvascular endothelial cells to the main inflammatory mediators involved in cytokine storms induced by sepsis and SARS-CoV2 pulmonary infection and to sera from healthy donors and severely ill patients with sepsis. Endothelial barrier function was measured by electric cell-substrate impedance sensing, quantitative confocal microscopy, and Western blot. RESULTS: The human lung microvascular endothelial cell barrier was completely disrupted by IL (interleukin)-6 conjugated with soluble IL-6R (IL-6 receptor) and by IL-1ß (interleukin-1beta), moderately affected by TNF (tumor necrosis factor)-α and IFN (interferon)-γ and unaffected by other cytokines and chemokines, such as IL-6, IL-8, MCP (monocyte chemoattractant protein)-1 and MCP-3. The inhibition of IL-1 and IL-6R simultaneously, but not separately, significantly reduced endothelial hyperpermeability on exposing human lung microvascular endothelial cells to a cytokine storm consisting of 8 inflammatory mediators or to sera from patients with sepsis. Simultaneous inhibition of IL-1 and JAK (Janus kinase)-STAT (signal transducer and activator of transcription protein), a signaling node downstream IL-6 and IFN-γ, also prevented septic serum-induced endothelial barrier disruption. CONCLUSIONS: These findings strongly suggest a major role for both IL-6 trans-signaling and IL-1ß signaling in the pathological increase in permeability of the human lung microvasculature and reveal combinatorial strategies that enable the gradual control of pulmonary endothelial barrier function in response to a cytokine storm.

The Role of the Gut Microbiota in Sanfilippo Syndrome's Physiopathology: An Approach in Two Affected Siblings.

Sanfilippo syndrome, or mucopolysaccharidosis type III (MPS III), is a rare lysosomal disease caused by congenital enzymatic deficiencies in heparan sulfate (HS) degradation, leading to organ dysfunction. The most severe hallmark of MPS III comprises neurological alterations, although gastrointestinal symptoms (GISs) have also been shown to be relevant in many patients. Here, we explored the contribution of the gut microbiota to MPS III GISs. We analyzed the composition and functionality of the gut microbiota in two MPS III siblings with the same mutation (c.544C > T, c.1080delC, in the SGSH gene) and the same diet, but with differences in their GISs, including recurrent diarrhea in one of them. Using 16S sequencing, we observed that the MPS III patients exhibited decreased alpha diversity and a lower abundance of Lachnospiraceae and Bifidobacteriaceae accompanied by a higher abundance of the Ruminococcaceae and Rikenellaceae families than the healthy control subjects. Comparing siblings, we found an increased abundance of Bacteroidaceae and a lower abundance of Ruminococcaceae and Akkermansiaceae in the GIS-free patient. This patient also had a higher relative abundance of Sus genes (SusA, SusB, SusE, and SusG) involved in glycosaminoglycan metabolism. We found higher HS levels in the stool of the two MPS III patients than in healthy volunteers, particularly in the patient with GISs. Functionally, whole fecal metabolites from the patient with GISs induced oxidative stress in vitro in healthy monocytes. Finally, the Bacteroides thetaiotaomicron strain isolated from MPS III stool samples exhibited HS degradation ability. Overall, our results reveal different microbiota compositions and functionalities in MPS III siblings, who exhibited differential gastrointestinal symptomatology. Our study may serve as a gateway to explore the impact of the gut microbiota and its potential to enhance the quality of life in Sanfilippo syndrome patients.

The Impact of Colistin Resistance on the Activation of Innate Immunity by Lipopolysaccharide Modification.

Colistin resistance is acquired by different lipopolysaccharide (LPS) modifications. We proposed to evaluate the of effect in vivo colistin resistance acquisition on the innate immune response. We used a pair of ST11 clone Klebsiella pneumoniae strains: an OXA-48, CTX-M-15 K. pneumoniae strain susceptible to colistin (CS-Kp) isolated from a urinary infection and its colistin-resistant variant (CR-Kp) from the same patient after prolonged treatment with colistin. No mutation of previously described genes for colistin resistance (pmrA, pmrB, mgrB, phoP/Q, arnA, arnC, arnT, ugdH, and crrAB) was found in the CR-Kp genome; however, LPS modifications were characterized by negative-ion matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. The strains were cocultured with human monocytes to determine their survival after phagocytosis and induction to apoptosis. Also, monocytes were stimulated with bacterial LPS to study cytokine and immune checkpoint production. The addition of 4-amino-4-deoxy-l-arabinose (Ara4N) to lipid A of CR-Kp accounted for the colistin resistance. CR-Kp survived significantly longer inside human monocytes after being phagocytosed than did the CS-Kp strain. In addition, LPS from CR-Kp induced both higher apoptosis in monocytes and higher levels of cytokine and immune checkpoint production than LPS from CS-Kp. Our data reveal a variable impact of colistin resistance on the innate immune system, depending on the responsible mechanism. Adding Ara4N to LPS in K. pneumoniae increases bacterial survival after phagocytosis and elicits a higher inflammatory response than its colistin-susceptible counterpart.

SARS-CoV-2 Proteins Induce Endotoxin Tolerance Hallmarks: A Demonstration in Patients with COVID-19.

According to a large number of reported cohorts, sepsis has been observed in nearly all deceased patients with COVID-19. We and others have described sepsis, among other pathologies, to be an endotoxin tolerance (ET)-related disease. In this study, we demonstrate that the culture of human blood cells from healthy volunteers in the presence of SARS-CoV-2 proteins induced ET hallmarks, including impairment of proinflammatory cytokine production, low MHC class II (HLA-DR) expression, poor T cell proliferation, and enhancing of both phagocytosis and tissue remodeling. Moreover, we report the presence of SARS-CoV-2 blood circulating proteins in patients with COVID-19 and how these levels correlate with an ET status, the viral RNA presence of SARS-CoV-2 in plasma, as well as with an increase in the proportion of patients with secondary infections.

High-fat diet activates splenic NOD1 and enhances neutrophil recruitment and neutrophil extracellular traps release in the spleen of ApoE-deficient mice.

In the course of atherogenesis, the spleen plays an important role in the regulation of extramedullary hematopoiesis, and in the control of circulating immune cells, which contributes to plaque progression. Here, we have investigated the role of splenic nucleotide-binding oligomerization domain 1 (NOD1) in the recruitment of circulating immune cells, as well as the involvement of this immune organ in extramedullary hematopoiesis in mice fed on a high-fat high-cholesterol diet (HFD). Under HFD conditions, the absence of NOD1 enhances the mobilization of immune cells, mainly neutrophils, from the bone marrow to the blood. To determine the effect of NOD1-dependent mobilization of immune cells under pro-atherogenic conditions, Apoe-/- and Apoe-/-Nod1-/- mice fed on HFD for 4 weeks were used. Splenic NOD1 from Apoe-/- mice was activated after feeding HFD as inferred by the phosphorylation of the NOD1 downstream targets RIPK2 and TAK1. Moreover, this activation was accompanied by the release of neutrophil extracellular traps (NETs), as determined by the increase in the expression of peptidyl arginine deiminase 4, and the identification of citrullinated histone H3 in this organ. This formation of NETs was significantly reduced in Apoe-/-Nod1-/- mice. Indeed, the presence of Ly6G+ cells and the lipidic content in the spleen of mice deficient in Apoe and Nod1 was reduced when compared to the Apoe-/- counterparts, which suggests that the mobilization and activation of circulating immune cells are altered in the absence of NOD1. Furthermore, confirming previous studies, Apoe-/-Nod1-/- mice showed a reduced atherogenic disease, and diminished recruitment of neutrophils in the spleen, compared to Apoe-/- mice. However, splenic artery ligation reduced the atherogenic burden in Apoe-/- mice an effect that, unexpectedly was lost in Apoe-/-Nod1-/- mice. Together, these results suggest that neutrophil accumulation and activity in the spleen are driven in part by NOD1 activation in mice fed on HFD, contributing in this way to regulating atherogenic progression.

Thiosulfinate-Enriched Allium sativum Extract Exhibits Differential Effects between Healthy and Sepsis Patients: The Implication of HIF-1α.

Garlic (Allium sativum) has historically been associated with antioxidant, immunomodulatory, and microbiocidal properties, mainly due to its richness in thiosulfates and sulfur-containing phytoconstituents. Sepsis patients could benefit from these properties because it involves both inflammatory and refractory processes. We evaluated the effects of thiosulfinate-enriched Allium sativum extract (TASE) on the immune response to bacterial lipopolysaccharide (LPS) by monocytes from healthy volunteers (HVs) and patients with sepsis. We also explored the TASE effects in HIF-1α, described as the key transcription factor leading to endotoxin tolerance in sepsis monocytes through IRAK-M expression. Our results showed TASE reduced the LPS-triggered reactive oxygen species (ROS) production in monocytes from both patients with sepsis and HVs. Moreover, this extract significantly reduced tumor necrosis factor (TNF)-α, interleukin-1ß, and interleukin-6 production in LPS-stimulated monocytes from HVs. However, TASE enhanced the inflammatory response in monocytes from patients with sepsis along with increased expression of human leukocyte antigen-DR. Curiously, these dual effects of TASE on immune response were also found when the HV cohort was divided into low- and high-LPS responders. Although TASE enhanced TNFα production in the LPS-low responders, it decreased the inflammatory response in the LPS-high responders. Furthermore, TASE decreased the HIF-1α pathway-associated genes IRAK-M, VEGFA and PD-L1 in sepsis cells, suggesting HIF-1α inhibition by TASE leads to higher cytokine production in these cells as a consequence of IRAK-M downregulation. The suppression of this pathway by TASE was confirmed in vitro with the prolyl hydroxylase inhibitor dimethyloxalylglycine. Our data revealed TASE's dual effect on monocyte response according to status/phenotype and suggested the HIF-1α suppression as the possible underlying mechanism.

Fused Cells between Human-Adipose-Derived Mesenchymal Stem Cells and Monocytes Keep Stemness Properties and Acquire High Mobility.

Human-adipose-derived mesenchymal stem cells (hADMSCs) are multipotent stem cells which have become of great interest in stem-cell therapy due to their less invasive isolation. However, they have limited migration and short lifespans. Therefore, understanding the mechanisms by which these cells could migrate is of critical importance for regenerative medicine. Methods: Looking for novel alternatives, herein, hADMSCs were isolated from adipose tissue and co-cultured with human monocytes ex vivo. Results: A new fused hybrid entity, a foam hybrid cell (FHC), which was CD90+CD14+, resulted from this co-culture and was observed to have enhanced motility, proliferation, immunomodulation properties, and maintained stemness features. Conclusions: Our study demonstrates the generation of a new hybrid cellular population that could provide migration advantages to MSCs, while at the same time maintaining stemness properties.

Circulating Monocytes Exhibit an Endotoxin Tolerance Status after Acute Ischemic Stroke: Mitochondrial DNA as a Putative Explanation for Poststroke Infections.

Patients with acute ischemic stroke (AIS) suffer from infections associated with mortality. The relevance of the innate immune system, and monocytes in particular, has emerged as an important factor in the evolution of these infections. The study enrolled 14 patients with AIS, without previous treatment, and 10 healthy controls. In the present study, we show that monocytes from patients with AIS exhibit a refractory state or endotoxin tolerance. The patients were unable to orchestrate an inflammatory response against LPS and expressed three factors reported to control the evolution of human monocytes into a refractory state: IL-1R-associated kinase-M, NFkB2/p100, and hypoxia-inducible factor-1α. The levels of circulating mitochondrial DNA (mtDNA) in patients with AIS correlated with impaired inflammatory response of isolated monocytes. Interestingly, the patients could be classified into two groups: those who were infected and those who were not, according to circulating mtDNA levels. This finding was validated in an independent cohort of 23 patients with AIS. Additionally, monocytes from healthy controls, cultured in the presence of both sera from patients and mtDNA, reproduced a refractory state after endotoxin challenge. This effect was negated by either a TLR9 antagonist or DNase treatment. The present data further extend our understanding of endotoxin tolerance implications in AIS. A putative role of mtDNA as a new biomarker of stroke-associated infections, and thus a clinical target for preventing poststroke infection, has also been identified.

Age-dependent hypoxia-induced PD-L1 upregulation in patients with obstructive sleep apnoea.

BACKGROUND AND OBJECTIVE: In obstructive sleep apnoea (OSA), intermittent hypoxia (IH) compromises immune surveillance through the upregulation of the programmed cell death-1 (PD-1) receptor and its ligand (PD-L1). Because the risk of OSA-related cancer depends on age, we assessed PD-L1/PD-1 expression in middle-aged and older patients with OSA as well as in a murine model. METHODS: PD-L1 expression was studied in 41 patients with severe OSA and 40 healthy volunteers (HV), divided into two groups (≤55 and >55 years of age). We used flow cytometry, quantitative PCR (qPCR) and ELISA to determine PD-L1 expression on monocytes and plasma PD-L1 protein levels. Moreover, we analysed PD-L1 expression on an in vivo IH model with old and young mice. RESULTS: In subjects up to 55 years of age, severe OSA increased PD-L1 surface protein and mRNA level expression on monocytes and soluble-PD-L1 protein concentration in plasma compared to HV. PD-L1 and hypoxia-induced factor (HIF)-1α expression correlated with age in HV, whereas in patients with OSA there was a negative relationship. In the mice exposed to IH, PD-L1 expression on F4/80+ splenocytes was also only increased in young animals. HIF-1α expression was significantly higher in patients with OSA than in HV in subjects up to 55 years of age, while PD-L1 expression in monocytes was related to HIF-1α expression in young patients with OSA. CONCLUSION: PD-L1 upregulation in patients with OSA as a consequence of HIF-1α activation occurs mainly in young patients. In older patients with OSA, upregulation was not detected, possibly due to impaired oxygen sensitivity.

PD-L1 Overexpression During Endotoxin Tolerance Impairs the Adaptive Immune Response in Septic Patients via HIF1α.

Sepsis, among other pathologies, is an endotoxin tolerance (ET)-related disease. On admission, we classified 48 patients with sepsis into 3 subgroups according to the ex vivo response to lipopolysaccharide. This response correlates with the Acute Physiology and Chronic Health Evaluation (APACHE) II score and the ET degree. Moreover, the ET-related classification determines the outcome of these patients. Programmed cell death-ligand 1 (PD-L1) expression on septic monocytes is also linked with ET status. In addition to the regulation of cytokine production, one of the hallmarks of ET that significantly affects patients with sepsis is T-cell proliferation impairment or a poor switch to the adaptive response. PD-L1/programmed cell death-1 (PD-1) blocking and knockdown assays on tolerant monocytes from both patients with sepsis and the in vitro model reverted the impaired adaptive response. Mechanistically, the transcription factor hypoxia-inducible factor-1α (HIF1α) has been translocated into the nucleus and drives PD-L1 expression during ET in human monocytes. This fact, together with patient classification according to the ex vivo lipopolysaccharide response, opens an interesting field of study and potential personalized clinical applications, not only for sepsis but also for all ET-associated pathologies.