RAGE engagement by SARS-CoV-2 enables monocyte infection and underlies COVID-19 severity.

The spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has fueled the COVID-19 pandemic with its enduring medical and socioeconomic challenges because of subsequent waves and long-term consequences of great concern. Here, we chart the molecular basis of COVID-19 pathogenesis by analyzing patients' immune responses at single-cell resolution across disease course and severity. This approach confirms cell subpopulation-specific dysregulation in COVID-19 across disease course and severity and identifies a severity-associated activation of the receptor for advanced glycation endproducts (RAGE) pathway in monocytes. In vitro THP1-based experiments indicate that monocytes bind the SARS-CoV-2 S1-receptor binding domain (RBD) via RAGE, pointing to RAGE-Spike interaction enabling monocyte infection. Thus, our results demonstrate that RAGE is a functional receptor of SARS-CoV-2 contributing to COVID-19 severity.

High-Throughput Microscopy Analysis of Mitochondrial Membrane Potential in 2D and 3D Models.

Recent proteomic, metabolomic, and transcriptomic studies have highlighted a connection between changes in mitochondria physiology and cellular pathophysiological mechanisms. Secondary assays to assess the function of these organelles appear fundamental to validate these -omics findings. Although mitochondrial membrane potential is widely recognized as an indicator of mitochondrial activity, high-content imaging-based approaches coupled to multiparametric to measure it have not been established yet. In this paper, we describe a methodology for the unbiased high-throughput quantification of mitochondrial membrane potential in vitro, which is suitable for 2D to 3D models. We successfully used our method to analyze mitochondrial membrane potential in monolayers of human fibroblasts, neural stem cells, spheroids, and isolated muscle fibers. Moreover, by combining automated image analysis and machine learning, we were able to discriminate melanoma cells from macrophages in co-culture and to analyze the subpopulations separately. Our data demonstrated that our method is a widely applicable strategy for large-scale profiling of mitochondrial activity.

Myonecrosis Induction by Intramuscular Injection of CTX.

Skeletal muscle, one of the most abundant tissue in the body, is a highly regenerative tissue. Indeed, compared to other tissues that are not able to regenerate after injury, skeletal muscle can fully regenerate upon mechanically, chemically, and infection-induced trauma. Several injury models have been developed to thoroughly investigate the physiological mechanisms regulating skeletal muscle regeneration. This protocol describes how to induce muscle regeneration by taking advantage of a cardiotoxin (CTX)-induced muscle injury model. The overall steps include CTX injection of tibialis anterior (TA) muscles of BL6N mice, collection of regenerating muscles at different time points after CTX injury, and histological characterization of regenerating muscles. Our protocol, compared with others such as those for freeze-induced injury models, avoids laceration or infections of the muscles since it involves neither surgery nor suture. In addition, our protocol is highly reproducible, since it causes homogenous myonecrosis of the whole muscle, and further reduces animal pain and stress. Graphical abstract.

OPA1 drives macrophage metabolism and functional commitment via p65 signaling.

Macrophages are essential players for the host response against pathogens, regulation of inflammation and tissue regeneration. The wide range of macrophage functions rely on their heterogeneity and plasticity that enable a dynamic adaptation of their responses according to the surrounding environmental cues. Recent studies suggest that metabolism provides synergistic support for macrophage activation and elicitation of desirable immune responses; however, the metabolic pathways orchestrating macrophage activation are still under scrutiny. Optic atrophy 1 (OPA1) is a mitochondria-shaping protein controlling mitochondrial fusion, cristae biogenesis and respiration; clear evidence shows that the lack or dysfunctional activity of this protein triggers the accumulation of metabolic intermediates of the TCA cycle. In this study, we show that OPA1 has a crucial role in macrophage activation. Selective Opa1 deletion in myeloid cells impairs M1-macrophage commitment. Mechanistically, Opa1 deletion leads to TCA cycle metabolite accumulation and defective NF-κB signaling activation. In an in vivo model of muscle regeneration upon injury, Opa1 knockout macrophages persist within the damaged tissue, leading to excess collagen deposition and impairment in muscle regeneration. Collectively, our data indicate that OPA1 is a key metabolic driver of macrophage functions.

Atypical CXCL12 signaling enhances neutrophil migration by modulating nuclear deformability.

To reach inflamed tissues from the circulation, neutrophils must overcome physical constraints imposed by the tissue architecture, such as the endothelial barrier or the three-dimensional (3D) interstitial space. In these microenvironments, neutrophils are forced to migrate through spaces smaller than their own diameter. One of the main challenges for cell passage through narrow gaps is the deformation of the nucleus, the largest and stiffest organelle in cells. Here, we showed that chemokines, the extracellular signals that guide cell migration in vivo, modulated nuclear plasticity to support neutrophil migration in restricted microenvironments. Exploiting microfabricated devices, we found that the CXC chemokine CXCL12 enhanced the nuclear pliability of mouse bone marrow-derived neutrophils to sustain their migration in 3D landscapes. This previously uncharacterized function of CXCL12 was mediated by the atypical chemokine receptor ACKR3 (also known as CXCR7), required protein kinase A (PKA) activity, and induced chromatin compaction, which resulted in enhanced cell migration in 3D. Thus, we propose that chemical cues regulate the nuclear plasticity of migrating leukocytes to optimize their motility in restricted microenvironments.

IL1ß Promotes TMPRSS2 Expression and SARS-CoV-2 Cell Entry Through the p38 MAPK-GATA2 Axis.

After the outburst of the SARS-CoV-2 pandemic, a worldwide research effort has led to the uncovering of many aspects of the COVID-19, among which we can count the outstanding role played by inflammatory cytokine milieu in the disease progression. Despite that, molecular mechanisms that regulate SARS-CoV-2 pathogenesis are still almost unidentified. In this study, we investigated whether the pro-inflammatory milieu of the host affects the susceptibility of SARS-CoV-2 infection by modulating ACE2 and TMPRSS2 expression. Our results indicated that the host inflammatory milieu favors SARS-CoV-2 infection by directly increasing TMPRSS2 expression. We unveiled the molecular mechanism that regulates this process and that can be therapeutically advantageously targeted.

The dominant-negative mitochondrial calcium uniporter subunit MCUb drives macrophage polarization during skeletal muscle regeneration.

Damaged skeletal muscle can regenerate because of the coordinated action of immune cells with muscle stem cells, called satellite cells. Proinflammatory macrophages infiltrate skeletal muscle soon after injury to sustain the proliferation of satellite cells. These macrophages later acquire the anti-inflammatory phenotype and promote the differentiation and fusion of satellite cells. Here, we showed that MCUb, the dominant-negative subunit of the mitochondrial calcium uniporter (MCU) complex, promotes muscle regeneration by controlling macrophage responses. Macrophages lacking MCUb lost the ability to efficiently acquire the anti-inflammatory profile, and mice with MCUb-deficient macrophages showed delayed regeneration through exhaustion of the satellite cell pool. MCUb ablation altered macrophage metabolism by promoting glycolysis and the accumulation of TCA cycle intermediates, which was accompanied by the stabilization of HIF-1α, the master transcriptional regulator of the macrophage proinflammatory program. Together, these data demonstrate that MCUb abundance is tightly controlled in macrophages to enable satellite cell functional differentiation and recovery of tissue homeostasis after damage.

The J2-Immortalized Murine Macrophage Cell Line Displays Phenotypical and Metabolic Features of Primary BMDMs in Their M1 and M2 Polarization State.

Macrophages are immune cells that are important for the development of the defensive front line of the innate immune system. Following signal recognition, macrophages undergo activation toward specific functional states, consisting not only in the acquisition of specific features but also of peculiar metabolic programs associated with each function. For these reasons, macrophages are often isolated from mice to perform cellular assays to study the mechanisms mediating immune cell activation. This requires expensive and time-consuming breeding and housing of mice strains. To overcome this issue, we analyzed an in-house J2-generated immortalized macrophage cell line from BMDMs, both from a functional and metabolic point of view. By assaying the intracellular and extracellular metabolism coupled with the phenotypic features of immortalized versus primary BMDMs, we concluded that classically and alternatively immortalized macrophages display similar phenotypical, metabolic and functional features compared to primary cells polarized in the same way. Our study validates the use of this immortalized cell line as a suitable model with which to evaluate in vitro how perturbations can influence the phenotypical and functional features of murine macrophages.

Age-severity matched cytokine profiling reveals specific signatures in Covid-19 patients.

A global effort is currently undertaken to restrain the COVID-19 pandemic. Host immunity has come out as a determinant for COVID-19 clinical outcomes, and several studies investigated the immune profiling of SARS-CoV-2 infected people to properly direct the clinical management of the disease. Thus, lymphopenia, T-cell exhaustion, and the increased levels of inflammatory mediators have been described in COVID-19 patients, in particular in severe cases1. Age represents a key factor in COVID-19 morbidity and mortality2. Understanding age-associated immune signatures of patients are therefore important to identify preventive and therapeutic strategies. In this study, we investigated the immune profile of COVID-19 hospitalized patients identifying a distinctive age-dependent immune signature associated with disease severity. Indeed, defined circulating factors - CXCL8, IL-10, IL-15, IL-27, and TNF-α - positively correlate with older age, longer hospitalization, and a more severe form of the disease and may thus represent the leading signature in critical COVID-19 patients.

Innate immunity ascertained from blood and tracheal aspirates of preterm newborn provides new clues for assessing bronchopulmonary dysplasia.

AIM: The study aimed to establish how granulocytes, monocytes and macrophages contribute to the development of bronchopulmonary dysplasia (BPD). MATERIALS AND METHODS: Study A: samples of blood and tracheal aspirates (TAs) collected from preterm newborn infants during the first 3 days of life were investigated by flow cytometry, and testing for white blood cells (WBCs), neutrophils and neutrophil extracellular traps (NETs). Maternal blood samples were also collected. Study B: data from previously-tested samples of TAs collected from preterm newborn infants were re-analyzed in the light of the findings in the new cohort. RESULTS: Study A: 39 preterm newborn infants were studied. A moderate correlation emerged between maternal WBCs and neutrophils and those of their newborn in the first 3 days of life. WBCs and neutrophils correlated in the newborn during the first 8 days of life. Decision rules based on birth weight (BW) and gestational age (GA) can be used to predict bronchopulmonary dysplasia (BPD). Neutrophil levels were lower in the TAs from the newborn with the lowest GAs and BWs. Study B: after removing the effect of GA on BPD development, previously-tested newborn were matched by GA. Monocyte phenotype 1 (Mon1) levels were lower in the blood of newborn with BPD, associated with a higher ratio of Monocyte phenotype 3 (Mon3) to Mon1. Newborn infants from mothers with histological chorioamnionitis (HCA) had lower levels of classically-activated macrophages (M1) and higher levels of alternatively-activated macrophages (M2) in their TAs than newborn infants from healthy mothers. CONCLUSION: Immune cell behavior in preterm newborn infants was examined in detail. Surprisingly, neutrophil levels were lower in TAs from the newborn with the lowest GA and BW, and no correlation emerged between the neutrophil and NET levels in TAs and the other variables measured. Interestingly, monocyte phenotype seemed to influence the onset of BPD. The rise in the ratio of Mon 3 to Mon 1 could contribute to endothelial dysfunction in BPD.

The Metabolic Signature of Macrophage Responses.

Macrophages are a heterogeneous population of immune cells playing several and diverse functions in homeostatic and immune responses. The broad spectrum of macrophage functions depends on both heterogeneity and plasticity of these cells, which are highly specialized in sensing the microenvironment and modify their properties accordingly. Although it is clear that macrophage phenotypes are difficult to categorize and should be seen as plastic and adaptable, they can be simplified into two extremes: a pro-inflammatory (M1) and an anti-inflammatory/pro-resolving (M2) profile. Based on this definition, M1 macrophages are able to start and sustain inflammatory responses, secreting pro-inflammatory cytokines, activating endothelial cells, and inducing the recruitment of other immune cells into the inflamed tissue; on the other hand, M2 macrophages promote the resolution of inflammation, phagocytose apoptotic cells, drive collagen deposition, coordinate tissue integrity, and release anti-inflammatory mediators. Dramatic switches in cell metabolism accompany these phenotypic and functional changes of macrophages. In particular, M1 macrophages rely mainly on glycolysis and present two breaks on the TCA cycle that result in accumulation of itaconate (a microbicide compound) and succinate. Excess of succinate leads to Hypoxia Inducible Factor 1α (HIF1α) stabilization that, in turn, activates the transcription of glycolytic genes, thus sustaining the glycolytic metabolism of M1 macrophages. On the contrary, M2 cells are more dependent on oxidative phosphorylation (OXPHOS), their TCA cycle is intact and provides the substrates for the complexes of the electron transport chain (ETC). Moreover, pro- and anti-inflammatory macrophages are characterized by specific pathways that regulate the metabolism of lipids and amino acids and affect their responses. All these metabolic adaptations are functional to support macrophage activities as well as to sustain their polarization in specific contexts. The aim of this review is to discuss recent findings linking macrophage functions and metabolism.

Intercellular Calcium Signaling Induced by ATP Potentiates Macrophage Phagocytosis.

Extracellular ATP is a signaling molecule exploited by the immune cells for both autocrine regulation and paracrine communication. By performing live calcium imaging experiments, we show that triggered mouse macrophages are able to propagate calcium signals to resting bystander cells by releasing ATP. ATP-based intercellular communication is mediated by P2X4 and P2X7 receptors and is a feature of pro-inflammatory macrophages. In terms of functional significance, ATP signaling is required for efficient phagocytosis of pathogen-derived molecules and apoptotic cells and may represent a target for macrophage regulation by CD39-expressing cells. These results highlight a cell-to-cell communication mechanism tuning innate immunity.

Helicobacter pylori Affects the Antigen Presentation Activity of Macrophages Modulating the Expression of the Immune Receptor CD300E through miR-4270.

Helicobacter pylori (Hp) is a Gram-negative bacterium that infects the human gastric mucosa, leading to chronic inflammation. If not eradicated with antibiotic treatment, the bacterium persists in the human stomach for decades increasing the risk to develop chronic gastritis, gastroduodenal ulcer, and gastric adenocarcinoma. The lifelong persistence of Hp in the human stomach suggests that the host response fails to clear the infection. It has been recently shown that during Hp infection phagocytic cells promote high Hp loads rather than contributing to bacterial clearance. Within these cells Hp survives in "megasomes," large structures arising from homotypic fusion of phagosomes, but the mechanism that Hp employs to avoid phagocytic killing is not completely understood. Here, we show that Hp infection induces the downregulation of specific microRNAs involved in the regulation of transcripts codifying for inflammatory proteins. miR-4270 targets the most upregulated gene: the immune receptor CD300E, whose expression is strictly dependent on Hp infection. CD300E engagement enhances the pro-inflammatory potential of macrophages, but in parallel it affects their ability to express and expose MHC class II molecules on the plasma membrane, without altering phagocytosis. This effect compromises the possibility for effector T cells to recognize and activate the killing potential of macrophages, which, in turn would become a survival niche for the bacterium. Taken together, our data add another piece to the complicate puzzle represented by the long-life coexistence between Hp and the human host and contribute with new insights toward understanding the regulation and function of the immune receptor CD300E.

A pH-sensitive stearoyl-PEG-poly(methacryloyl sulfadimethoxine)-decorated liposome system for protein delivery: An application for bladder cancer treatment.

Stealth pH-responsive liposomes for the delivery of therapeutic proteins to the bladder epithelium were prepared using methoxy-poly(ethylene glycol)5kDa-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (mPEG5kDa-DSPE) and stearoyl-poly(ethylene glycol)-poly(methacryloyl sulfadimethoxine) copolymer (stearoyl-PEG-polySDM), which possesses an apparent pKa of 7.2. Liposomes of 0.2:0.6:100, 0.5:1.5:100 and 1:3:100 mPEG5kDa-DSPE/stearoyl-PEG-polySDM/(soybean phosphatidylcholine+cholesterol) molar ratios were loaded with bovine serum albumin (BSA) as a protein model. The loading capacity was 1.3% w/w BSA/lipid. At pH7.4, all liposome formulations displayed a negative zeta-potential and were stable for several days. By pH decrease or addition to mouse urine, the zeta potential strongly decreased, and the liposomes underwent a rapid size increase and aggregation. Photon correlation spectroscopy (PCS) and transmission electron microscopy (TEM) analyses showed that the extent of the aggregation depended on the stearoyl-PEG-polySDM/lipid molar ratio. Cytofluorimetric analysis and confocal microscopy showed that at pH6.5, the incubation of MB49 mouse bladder cancer cells and macrophages with fluorescein isothiocyanate-labelled-BSA (FITC-BSA) loaded and N-(Lissamine Rhodamine B sulfonyl)-1, 2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine triethylammonium salt (rhodamine-DHPE) labelled 1:3:100 mPEG5kDa-DSPE/stearoyl-PEG-polySDM/lipid molar ratio liposomes resulted in a time-dependent liposome association with the cells. At pH7.4, the association of BSA-loaded liposomes with the MB49 cells and macrophages was remarkably lower than at pH6.5. Confocal images of bladder sections revealed that 2h after the instillation, liposomes at pH7.4 and control non-responsive liposomes at pH7.4 or 6.5 did not associate nor delivered FITC-BSA to the bladder epithelium. On the contrary, the pH-responsive liposome formulation set at pH6.5 and soon administered to mice by bladder instillation showed that, 2h after administration, the pH-responsive liposomes efficiently delivered the loaded FITC-BSA to the bladder epithelium.

Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model.

Bladder cancer is one of the most common malignancies of the urogenital tract. Intravesical injection of Bacillus Calmette-Guérin (BCG) is the gold standard treatment for the high-grade non-muscle invasive bladder cancer (NMIBC). However, since the treatment-related side effects are relevant, newer biological response modifiers with a better benefit/side effects ratio are needed. The tumour microenvironment can influence both tumour development and therapy efficacy. In order to obtain a good model, it is desirable to implant tumour cells in the organ from which the cancer originates. In this protocol, we describe a method for establishing a tumour in the bladder cavity of female mice and subsequent delivery of therapeutic agents; the latter are exemplified by our use of Helicobacter pylori neutrophil activating protein (HP-NAP). A preliminary chemical burn of the mucosa, followed by the injection of mouse urothelial carcinoma cell line MB49 via urethral catheterization, enables the cells to attach to the bladder mucosa. After a period, required to allow an initial proliferation of the cells, mice are treated with HP-NAP, administrated again via catheterization. The anti-tumour activity of HP-NAP is evaluated comparing the tumour volume, the extent of necrosis and the degree of vascularization between vehicle- and HP-NAP-treated animals.

Cytokine BAFF released by Helicobacter pylori-infected macrophages triggers the Th17 response in human chronic gastritis.

BAFF is a crucial cytokine that affects the activity of both innate and adaptive immune cells. It promotes the expansion of Th17 cells in autoimmune disorders. With this study, we investigated the BAFF/Th17 responses in Helicobacter pylori-induced gastritis in humans. Our results show that the mucosa from Helicobacter(+) patients with chronic gastritis is enriched in IL-17 and BAFF, whereas the two cytokines are weakly expressed in Helicobacter(-) patients with chronic gastritis; moreover, the expression of both BAFF and IL-17 decreases after bacteria eradication. We demonstrate that BAFF accumulates in macrophages in vivo and that it is produced by monocyte-derived macrophages in vitro, after Helicobacter stimulation. Application of BAFF on monocytes triggers the accumulation of reactive oxygen species that are crucial for the release of pro-Th17 cytokines, such as IL-23, IL-1ß, and TGF-ß. Moreover, BAFF directly promotes the differentiation of Th17 cells. In conclusion, our results support the notion that an axis BAFF/Th17 exists in chronic gastritis of Helicobacter(+) patients and that its presence strictly depends on the bacterium. Moreover, we demonstrated that BAFF is able to drive Th17 responses both indirectly, by creating a pro-Th17 cytokine milieu through the involvement of innate immune cells, and directly, via the differentiation of T cells toward the specific profile. The results obtained in this study are of great interest for Helicobacter-related diseases and the development of novel therapeutic strategies based on the inhibition of the BAFF/IL-17 response.

Helicobacter pylori secreted peptidyl prolyl cis, trans-isomerase drives Th17 inflammation in gastric adenocarcinoma.

Helicobacter pylori infection is characterized by an inflammatory infiltrate, consisting mainly of neutrophils and T cells. This study was undertaken to evaluate the type of gastric T cell response elicited by the secreted peptidyl prolyl cis, trans-isomerase of H. pylori (HP0175) in patients with distal gastric adenocarcinoma. The cytokine profile and the effector functions of gastric tumor-infiltrating lymphocytes (TILs) specific for HP0175 was investigated in 20 patients with distal gastric adenocarcinoma and H. pylori infection. The helper function of HP0175-specific TILs for monocyte MMP-2, MMP-9, and VEGF production was also investigated. TILs cells from H. pylori infected patients with distal gastric adenocarcinoma produced Interleukin (IL)-17 and IL-21 in response to HP0175. HP0175-specific TILs showed poor cytolytic activity while expressing helper activity for monocyte MMP-2, MMP-9 and VEGF production. These findings indicate that HP0175 is able to drive gastric Th17 response. Thus, HP0175, by promoting pro-inflammatory low cytotoxic TIL response, matrix degradation and pro-angiogenic pathways, may provide a link between H. pylori and gastric cancer.

[Therapy for non-muscle invasive bladder cancer: HP-NAP]. / La risposta immunitaria Th1 mediata nella terapia della neoplasia vescicale non muscolo invasiva: il ruolo di HP-NAP.

PURPOSE: Patients with non-muscle invasive bladder cancer recurrence after 2 induction courses of BCG are eligible for radical cystectomy. So, in the last years research to discover new drugs for the management of non-muscle invasive bladder cancer recurrence after failure of first and second line therapy is ongoing. In accordance to the results obtained with BCG, whose mechanism depends on the induction of the T helper 1 (TH1) immune response, we investigated the activity of a Toll-like receptor (TLR) 2 ligand, named Helicobacter Pylori Neutrophil Activating Protein (HP-NAP), that we recently demonstrated being able of enhancing the differentiation of Th1 cells, both in vitro and in vivo, because of its ability to create an IL-12 enriched milieu. MATERIALS AND METHODS: We show here, in a mouse model of bladder neoplasm implants, that local administration of HP-NAP decreases tumor growth by inducing tumor necrosis. RESULTS: The result is joined up with a massive cluster of both CD4+ and CD8+ IFN-γ+ cells, within neoplasm and regional lymph nodes. It is of note that HP-NAP-treated tumors show also a reduced vascularization due to the anti-angiogenic activity of IFN-γ induced by HP-NAP. CONCLUSIONS: The present study suggests that the activity of HP-NAP against urothelial tumor burden warrants subsequent in vivo studies.

Chlamydophila pneumoniae phospholipase D (CpPLD) drives Th17 inflammation in human atherosclerosis.

Phospholipases are produced from bacterial pathogens causing very different diseases. One of the most intriguing aspects of phospholipases is their potential to interfere with cellular signaling cascades and to modulate the host-immune response. Here, we investigated the role of the innate and acquired immune responses elicited by Chlamydophila pneumoniae phospholipase D (CpPLD) in the pathogenesis of atherosclerosis. We evaluated the cytokine and chemokine production induced by CpPLD in healthy donors' monocytes and in vivo activated T cells specific for CpPLD that infiltrate atherosclerotic lesions of patients with C. pneumoniae antibodies. We also examined the helper function of CpPLD-specific T cells for monocyte matrix metalloproteinase (MMP)-9 and tissue factor (TF) production as well as the CpPLD-induced chemokine expression by human venular endothelial cells (HUVECs). We report here that CpPLD is a TLR4 agonist able to induce the expression of IL-23, IL-6, IL-1ß, TGF-ß, and CCL-20 in monocytes, as well as CXCL-9, CCL-20, CCL-4, CCL-2, ICAM-1, and VCAM-1 in HUVECs. Plaque-derived T cells produce IL-17 in response to CpPLD. Moreover, CpPLD-specific CD4(+) T lymphocytes display helper function for monocyte MMP-9 and TF production. CpPLD promotes Th17 cell migration through the induction of chemokine secretion and adhesion molecule expression on endothelial cells. These findings indicate that CpPLD is able to drive the expression of IL-23, IL-6, IL-1ß, TGF-ß, and CCL-20 by monocytes and to elicit a Th17 immune response that plays a key role in the genesis of atherosclerosis.