Inactivated whole virion vaccine protects K18-hACE2 Tg mice against the Omicron SARS-CoV-2 variant via cross-reactive T cells and nonneutralizing antibody responses.

COVID-19 is a systemic inflammatory disease initiated by SARS-CoV-2 virus infection. Multiple vaccines against the Wuhan variant of SARS-CoV-2 have been developed including a whole virion beta-propiolactone-inactivated vaccine based on the B.1.1 strain (CoviVac). Since most of the population has been vaccinated by targeting the original or early variants of SARS-CoV-2, the emergence of novel mutant variants raises concern over possible evasion of vaccine-induced immune responses. Here, we report on the mechanism of protection by CoviVac, a whole virion-based vaccine, against the Omicron variant. CoviVac-immunized K18-hACE2 Tg mice were protected against both prototype B.1.1 and BA.1-like (Omicron) variants. Subsequently, vaccinated K18-hACE2 Tg mice rapidly cleared the infection via cross-reactive T-cell responses and cross-reactive, non-neutralizing antibodies recognizing the Omicron variant Spike protein. Thus, our data indicate that efficient protection from SARS-CoV-2 variants can be achieved by the orchestrated action of cross-reactive T cells and non-neutralizing antibodies.

Akkermansia muciniphila - friend or foe in colorectal cancer?

Akkermansia muciniphila is a gram-negative anaerobic bacterium, which represents a part of the commensal human microbiota. Decline in the abundance of A. muciniphila among other microbial species in the gut correlates with severe systemic diseases such as diabetes, obesity, intestinal inflammation and colorectal cancer. Due to its mucin-reducing and immunomodulatory properties, the use of probiotics containing Akkermansia sp. appears as a promising approach to the treatment of metabolic and inflammatory diseases. In particular, a number of studies have focused on the role of A. muciniphila in colorectal cancer. Of note, the results of these studies in mice are contradictory: some reported a protective role of A. muciniphila in colorectal cancer, while others demonstrated that administration of A. muciniphila could aggravate the course of the disease resulting in increased tumor burden. More recent studies suggested the immunomodulatory effect of certain unique surface antigens of A. muciniphila on the intestinal immune system. In this Perspective, we attempt to explain how A. muciniphila contributes to protection against colorectal cancer in some models, while being pathogenic in others. We argue that differences in the experimental protocols of administration of A. muciniphila, as well as viability of bacteria, may significantly affect the results. In addition, we hypothesize that antigens presented by pasteurized bacteria or live A. muciniphila may exert distinct effects on the barrier functions of the gut. Finally, A. muciniphila may reduce the mucin barrier and exerts combined effects with other bacterial species in either promoting or inhibiting cancer development.

Cardiomyocyte and endothelial cells play distinct roles in the tumour necrosis factor (TNF)-dependent atrial responses and increased atrial fibrillation vulnerability induced by endurance exercise training in mice.

AIMS: Endurance exercise is associated with an increased risk of atrial fibrillation (AF). We previously established that adverse atrial remodelling and AF susceptibility induced by intense exercise in mice require the mechanosensitive and pro-inflammatory cytokine tumour necrosis factor (TNF). The cellular and mechanistic basis for these TNF-mediated effects is unknown. METHODS AND RESULTS: We studied the impact of Tnf excision, in either atrial cardiomyocytes or endothelial cells (using Cre-recombinase expression controlled by Nppa or Tie2 promoters, respectively), on the cardiac responses to six weeks of intense swim exercise training. TNF ablation, in either cell type, had no impact on the changes in heart rate, autonomic tone, or left ventricular structure and function induced by exercise training. Tnf excision in atrial cardiomyocytes did, however, prevent atrial hypertrophy, fibrosis, and macrophage infiltration as well as conduction slowing and increased AF susceptibility arising from exercise training. In contrast, endothelial-specific excision only reduced the training-induced atrial hypertrophy. Consistent with these cell-specific effects of Tnf excision, inducing TNF loss from atrial cardiomyocytes prevented activation of p38MAPKinase, a strain-dependent downstream mediator of TNF signalling, without affecting the atrial stretch as assessed by atrial pressures induced by exercise. Despite TNF's established role in innate immune responses and inflammation, neither acute nor chronic exercise training caused measurable NLRP3 inflammasome activation. CONCLUSIONS: Our findings demonstrate that adverse atrial remodelling and AF vulnerability induced by intense exercise require TNF in atrial cardiomyocytes whereas the impact of endothelial-derived TNF is limited to hypertrophy modulation. The implications of the cell autonomous effects of TNF and crosstalk between cells in the atria are discussed.

Macrophages from naked mole-rat possess distinct immunometabolic signatures upon polarization.

The naked mole-rat (NMR) is a unique long-lived rodent which is highly resistant to age-associated disorders and cancer. The immune system of NMR possesses a distinct cellular composition with the prevalence of myeloid cells. Thus, the detailed phenotypical and functional assessment of NMR myeloid cell compartment may uncover novel mechanisms of immunoregulation and healthy aging. In this study gene expression signatures, reactive nitrogen species and cytokine production, as well as metabolic activity of classically (M1) and alternatively (M2) activated NMR bone marrow-derived macrophages (BMDM) were examined. Polarization of NMR macrophages under pro-inflammatory conditions led to expected M1 phenotype characterized by increased pro-inflammatory gene expression, cytokine production and aerobic glycolysis, but paralleled by reduced production of nitric oxide (NO). Under systemic LPS-induced inflammatory conditions NO production also was not detected in NMR blood monocytes. Altogether, our results indicate that NMR macrophages are capable of transcriptional and metabolic reprogramming under polarizing stimuli, however, NMR M1 possesses species-specific signatures as compared to murine M1, implicating distinct adaptations in NMR immune system.

SARS-CoV-2 Binding and Neutralization Properties of Peptides Derived from N-Terminus of Human ACE2.

The binding properties of synthetic and recombinant peptides derived from N-terminal part of ACE2, the main receptor for SARS-CoV-2, were evaluated. Additionally, the ability of these peptides to prevent virus entry in vitro was addressed using both pseudovirus particles decorated with the S protein, as well as through infection of Vero cells with live SARS-CoV-2 virus. Surprisingly, in spite of effective binding to S protein, all linear peptides of various lengths failed to neutralize the viral infection in vitro. However, the P1st peptide that was chemically "stapled" in order to stabilize its alpha-helical structure was able to interfere with virus entry into ACE2-expressing cells. Interestingly, this peptide also neutralized pseudovirus particles decorated with S protein derived from the Omicron BA.1 virus, in spite of variations in key amino acid residues contacting ACE2.

Will Peptides Help to Stop COVID-19?

Peptides are widely used for the diagnostics, prevention, and therapy of certain human diseases. How useful can they be for the disease caused by the SARS-CoV-2 coronavirus? In this review, we discuss the possibility of using synthetic and recombinant peptides and polypeptides for prevention of COVID-19 via blocking the interaction between the virus and its main receptor ACE2, as well as components of antiviral vaccines, in particular, against new emerging virus variants.

HDM induces distinct immunometabolic phenotype in macrophages in TLR4-dependent manner.

Asthma is one of the most common chronic diseases. In many cases it is preceded by the development of an immune response to allergens such as animal fur, dust, pollens and etc. In human population this disease is heterogeneous, and no selective drugs are available at the moment for some endotypes of asthma. The role of the adaptive immune system in the pathogenesis of asthma was extensively studied, while the role of innate immune cells, in particular myeloid cells, was not sufficiently addressed. Myeloid cells, such as macrophages and dendritic cells, are characterized by high plasticity, heterogenicity and ability to undergo polarization in response to various pathogenic stimuli, including those engaging innate immune receptors. Recently, special attention was drawn to the link between polarization of macrophages and cell metabolism. We hypothesized that immunometabolic reprogramming of myeloid cells, in particular, of macrophages and dendritic cells during sensitization with an allergen may affect further immune response and asthma development. To test this hypothesis, we generated distinct types of myeloid cells in vitro from murine bone marrow and analyzed their immunometabolic profiles upon activation with house dust mite extract (HDM) and its key active components. We found that the combination of lipopolysaccharide (LPS) and beta-glucan is sufficient to upregulate proinflammatory cytokine production as well as respiratory and glycolytic capacity of myeloid cells, comparably to HDM. This specific immunometabolic phenotype was associated with altered mitochondrial morphology and possibly with increased ROS production in macrophages. Moreover, we found that both TNF production and metabolic remodeling of macrophages in response to HDM are TLR4-dependent processes. Altogether, these results expand our understanding of molecular mechanisms underlying asthma induction and pathogenesis and may potentially lead to new therapeutic strategies for the treatment of this disease.

LTα, TNF, and ILC3 in Peyer's Patch Organogenesis.

TNF and LTα are structurally related cytokines of the TNF superfamily. Their genes are located in close proximity to each other and to the Ltb gene within the TNF/LT locus inside MHC. Unlike Ltb, transcription of Tnf and of Lta is tightly controlled, with the Tnf gene being an immediate early gene that is rapidly induced in response to various inflammatory stimuli. Genes of the TNF/LT locus play a crucial role in lymphoid tissue organogenesis, although some aspects of their specific contribution remain controversial. Here, we present new findings and discuss the distinct contribution of TNF produced by ILC3 cells to Peyer's patch organogenesis.

TNF hampers intestinal tissue repair in colitis by restricting IL-22 bioavailability.

Successful treatment of chronic inflammatory diseases integrates both the cessation of inflammation and the induction of adequate tissue repair processes. Strikingly, targeting a single proinflammatory cytokine, tumor necrosis factor (TNF), induces both processes in a relevant cohort of inflammatory bowel disease (IBD) patients. However, the molecular mechanisms underlying intestinal repair following TNF blockade during IBD remain elusive. Using a novel humanized model of experimental colitis, we demonstrate that TNF interfered with the tissue repair program via induction of a soluble natural antagonist of IL-22 (IL-22Ra2; IL-22BP) in the colon and abrogated IL-22/STAT3-mediated mucosal repair during colitis. Furthermore, membrane-bound TNF expressed by T cells perpetuated colonic inflammation, while soluble TNF produced by epithelial cells (IECs) induced IL-22BP expression in colonic dendritic cells (DCs) and dampened IL-22-driven restitution of colonic epithelial functions. Finally, TNF induced IL-22BP expression in human monocyte-derived DCs and levels of IL22-BP correlated with TNF in sera of IBD patients. Thus, our data can explain how anti-TNF therapy induces mucosal healing by increasing IL-22 availability and implicates new therapeutic opportunities for IBD.

Therapeutic Potential of Combining IL-6 and TNF Blockade in a Mouse Model of Allergic Asthma.

Combined anti-cytokine therapy is a promising therapeutic approach for uncontrolled steroid-resistant asthma. In this regard, simultaneous blockade of IL-4 and IL-13 signaling by Dupilumab (anti-IL-4Ra monoclonal antibody) was recently approved for severe eosinophilic asthma. However, no therapeutic options for neutrophilic asthma are currently available. Recent advances in our understanding of asthma pathogenesis suggest that both IL-6 and TNF may represent potential targets for treatment of severe neutrophilic asthma. Nevertheless, the efficacy of simultaneous pharmacological inhibition of TNF and IL-6 in asthma was not yet studied. To evaluate the potency of combined cytokine inhibition, we simultaneously administrated IL-6 and TNF inhibitors to BALB/c mice with HDM-induced asthma. Combined IL-6/TNF inhibition, but not individual blockade of these two cytokines, led to complex anti-inflammatory effects including reduced Th2-induced eosinophilia and less prominent Th17/Th1-mediated neutrophilic infiltrate in the airways. Taken together, our results provide evidence for therapeutic potential of combined IL-6/TNF inhibition in severe steroid-resistant asthma.

Macrophages acquire a TNF-dependent inflammatory memory in allergic asthma.

BACKGROUND: Infectious agents can reprogram or "train" macrophages and their progenitors to respond more readily to subsequent insults. However, whether such an inflammatory memory exists in type 2 inflammatory conditions such as allergic asthma was not known. OBJECTIVE: We sought to decipher macrophage-trained immunity in allergic asthma. METHODS: We used a combination of clinical sampling of house dust mite (HDM)-allergic patients, HDM-induced allergic airway inflammation in mice, and an in vitro training setup to analyze persistent changes in macrophage eicosanoid, cytokine, and chemokine production as well as the underlying metabolic and epigenetic mechanisms. Transcriptional and metabolic profiles of patient-derived and in vitro trained macrophages were assessed by RNA sequencing or metabolic flux analysis and liquid chromatography-tandem mass spectrometry analysis, respectively. RESULTS: We found that macrophages differentiated from bone marrow or blood monocyte progenitors of HDM-allergic mice or asthma patients show inflammatory transcriptional reprogramming and excessive mediator (TNF-α, CCL17, leukotriene, PGE2, IL-6) responses upon stimulation. Macrophages from HDM-allergic mice initially exhibited a type 2 imprint, which shifted toward a classical inflammatory training over time. HDM-induced allergic airway inflammation elicited a metabolically activated macrophage phenotype, producing high amounts of 2-hydroxyglutarate (2-HG). HDM-induced macrophage training in vitro was mediated by a formyl peptide receptor 2-TNF-2-HG-PGE2/PGE2 receptor 2 axis, resulting in an M2-like macrophage phenotype with high CCL17 production. TNF blockade by etanercept or genetic ablation of Tnf in myeloid cells prevented the inflammatory imprinting of bone marrow-derived macrophages from HDM-allergic mice. CONCLUSION: Allergen-triggered inflammation drives a TNF-dependent innate memory, which may perpetuate and exacerbate chronic type 2 airway inflammation and thus represents a target for asthma therapy.

Novel Anti-Cytokine Strategies for Prevention and Treatment of Respiratory Allergic Diseases.

Asthma is a heterogeneous inflammatory disease characterized by airflow obstruction, wheezing, eosinophilia and neutrophilia of the airways. Identification of distinct inflammatory patterns characterizing asthma endotypes led to the development of novel therapeutic approaches. Cytokine or cytokine receptor targeting by therapeutic antibodies, such as anti-IL-4 and anti-IL-5, is now approved for severe asthma treatment. However, the complexity of cytokine networks in asthma should not be underestimated. Inhibition of one pro-inflammatory cytokine may lead to perturbed expression of another pro-inflammatory cytokine. Without understanding of the underlying mechanisms and defining the molecular predictors it may be difficult to control cytokine release that accompanies certain disease manifestations. Accumulating evidence suggests that in some cases a combined pharmacological inhibition of pathogenic cytokines, such as simultaneous blockade of IL-4 and IL-13 signaling, or blockade of upstream cytokines, such as TSLP, are more effective than single cytokine targeting. IL-6 and TNF are the important inflammatory mediators in the pathogenesis of asthma. Preliminary data suggests that combined pharmacological inhibition of TNF and IL-6 during asthma may be more efficient as compared to individual neutralization of these cytokines. Here we summarize recent findings in the field of anti-cytokine therapy of asthma and discuss immunological mechanisms by which simultaneous targeting of multiple cytokines as opposed to targeting of a single cytokine may improve disease outcomes.

Current Perspectives on the Role of TNF in Hematopoiesis Using Mice With Humanization of TNF/LT System.

TNF is a multifunctional cytokine with its key functions attributed to inflammation, secondary lymphoid tissue organogenesis and immune regulation. However, it is also a physiological regulator of hematopoiesis and is involved in development and homeostatic maintenance of various organs and tissues. Somewhat unexpectedly, the most important practical application of TNF biology in medicine is anti-TNF therapy in several autoimmune diseases. With increased number of patients undergoing treatment with TNF inhibitors and concerns regarding possible adverse effects of systemic cytokine blockade, the interest in using humanized mouse models to study the efficacy and safety of TNF-targeting biologics in vivo is justified. This Perspective discusses the main functions of TNF and its two receptors, TNFR1 and TNFR2, in steady state, as well as in emergency hematopoiesis. It also provides a comparative overview of existing mouse lines with humanization of TNF/TNFR system. These genetically engineered mice allow us to study TNF signaling cascades in the hematopoietic compartment in the context of various experimental disease models and for evaluating the effects of various human TNF inhibitors on hematopoiesis and other physiological processes.

Dual Role of TNF and LTα in Carcinogenesis as Implicated by Studies in Mice.

Tumor necrosis factor (TNF) and lymphotoxin alpha (LTα) are two related cytokines from the TNF superfamily, yet they mediate their functions in soluble and membrane-bound forms via overlapping, as well as distinct, molecular pathways. Their genes are encoded within the major histocompatibility complex class III cluster in close proximity to each other. TNF is involved in host defense, maintenance of lymphoid tissues, regulation of cell death and survival, and antiviral and antibacterial responses. LTα, known for some time as TNFß, has pleiotropic functions including control of lymphoid tissue development and homeostasis cross talk between lymphocytes and their environment, as well as lymphoid tissue neogenesis with formation of lymphoid follicles outside the lymph nodes. Along with their homeostatic functions, deregulation of these two cytokines may be associated with initiation and progression of chronic inflammation, autoimmunity, and tumorigenesis. In this review, we summarize the current state of knowledge concerning TNF/LTα functions in tumor promotion and suppression, with the focus on the recently uncovered significance of host-microbiota interplay in cancer development that may explain some earlier controversial results.

Fibroblasts upregulate expression of adhesion molecules and promote lymphocyte retention in 3D fibroin/gelatin scaffolds.

Bioengineered scaffolds are crucial components in artificial tissue construction. In general, these scaffolds provide inert three-dimensional (3D) surfaces supporting cell growth. However, some scaffolds can affect the phenotype of cultured cells, especially, adherent stromal cells, such as fibroblasts. Here we report on unique properties of 3D fibroin/gelatin materials, which may rapidly induce expression of adhesion molecules, such as ICAM-1 and VCAM-1, in cultured primary murine embryonic fibroblasts (MEFs). In contrast, two-dimensional (2D) fibroin/gelatin films did not show significant effects on gene expression profiles in fibroblasts as compared to 3D culture conditions. Interestingly, TNF expression was induced in MEFs cultured in 3D fibroin/gelatin scaffolds, while genetic or pharmacological TNF ablation resulted in diminished ICAM-1 and VCAM-1 expression by these cells. Using selective MAPK inhibitors, we uncovered critical contribution of JNK to 3D-induced upregulation of these adhesion molecules. Moreover, we observed ICAM-1/VCAM-1-dependent adhesion of lymphocytes to fibroblasts cultured in 3D fibroin/gelatin scaffolds, but not on 2D fibroin/gelatin films, suggesting functional reprogramming in stromal cells, when exposed to 3D environment. Finally, we observed significant infiltration of lymphocytes into 3D fibroin/gelatin, but not into collagen scaffolds in vivo upon subcapsular kidney implantation in mice. Together our data highlight the important features of fibroin/gelatin scaffolds, when they are produced as 3D sponges rather than 2D films, which should be considered when using these materials for tissue engineering.

Directional mast cell degranulation of tumor necrosis factor into blood vessels primes neutrophil extravasation.

Tissue resident mast cells (MCs) rapidly initiate neutrophil infiltration upon inflammatory insult, yet the molecular mechanism is still unknown. Here, we demonstrated that MC-derived tumor necrosis factor (TNF) was crucial for neutrophil extravasation to sites of contact hypersensitivity-induced skin inflammation by promoting intraluminal crawling. MC-derived TNF directly primed circulating neutrophils via TNF receptor-1 (TNFR1) while being dispensable for endothelial cell activation. The MC-derived TNF was infused into the bloodstream by directional degranulation of perivascular MCs that were part of the vascular unit with access to the vessel lumen. Consistently, intravenous administration of MC granules boosted neutrophil extravasation. Pronounced and rapid intravascular MC degranulation was also observed upon IgE crosslinking or LPs challenge indicating a universal MC potential. Consequently, the directional MC degranulation of pro-inflammatory mediators into the bloodstream may represent an important target for therapeutic approaches aimed at dampening cytokine storm syndromes or shock symptoms, or intentionally pushing immune defense.

Conditional Ablation of Myeloid TNF Improves Functional Outcome and Decreases Lesion Size after Spinal Cord Injury in Mice.

Spinal cord injury (SCI) is a devastating condition consisting of an instant primary mechanical injury followed by a secondary injury that progresses for weeks to months. The cytokine tumor necrosis factor (TNF) plays an important role in the pathophysiology of SCI. We investigated the effect of myeloid TNF ablation (peripheral myeloid cells (macrophages and neutrophils) and microglia) versus central myeloid TNF ablation (microglia) in a SCI contusion model. We show that TNF ablation in macrophages and neutrophils leads to reduced lesion volume and improved functional outcome after SCI. In contrast, TNF ablation in microglia only or TNF deficiency in all cells had no effect. TNF levels tended to be decreased 3 h post-SCI in mice with peripheral myeloid TNF ablation and was significantly decreased 3 days after SCI. Leukocyte and microglia populations and all other cytokines (IL-1ß, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, and IFNγ) and chemokines (CCL2, CCL5, and CXCL1) investigated, in addition to TNFR1 and TNFR2, were comparable between genotypes. Analysis of post-SCI signaling cascades demonstrated that the MAPK kinase SAPK/JNK decreased and neuronal Bcl-XL levels increased post-SCI in mice with ablation of TNF in peripheral myeloid cells. These findings demonstrate that peripheral myeloid cell-derived TNF is pathogenic in SCI.

The structure of myeloid cell-specific TNF inhibitors affects their biological properties.

Spatial organization and conformational changes of antibodies may significantly affect their biological functions. We assessed the effect of mutual organization of the two VH H domains within bispecific antibodies recognizing human TNF and the surface molecules of murine myeloid cells (F4/80 or CD11b) on TNF retention and inhibition. TNF-neutralizing properties in vitro and in vivo of MYSTI-2 and MYSTI-3 antibodies were compared with new variants with interchanged VH H domains and different linker sequences. The most effective structure of MYSTI-2 and MYSTI-3 proteins required the Ser/Gly-containing 'superflexible' linker. The orientation of the modules was crucial for the activity of the proteins, but not for MYSTI-3 with the Pro/Gln-containing 'semi-rigid' linker. Our results may contribute toward the development of more effective drug prototypes.

Experimental Subarachnoid Hemorrhage Drives Catecholamine-Dependent Cardiac and Peripheral Microvascular Dysfunction.

Subarachnoid hemorrhage (SAH) is a devastating cerebral event caused by an aneurysmal rupture. In addition to neurological injury, SAH has significant effects on cardiac function and the peripheral microcirculation. Since these peripheral complications may exacerbate brain injury, the prevention and management of these peripheral effects are important for improving the overall clinical outcome after SAH. In this investigation, we examined the effects of SAH on cardiac function and vascular reactivity in a well-characterized blood injection model of SAH. Standard echocardiographic and blood pressure measurement procedures were utilized to assess cardiac function and hemodynamic parameters in vivo; we utilized a pressure myography approach to assess vascular reactivity in cremaster skeletal muscle resistance arteries ex vivo. We observed that elevated catecholamine levels in SAH stun the myocardium, reduce cardiac output and augment myogenic vasoconstriction in isolated cremaster arteries. These cardiac and vascular effects are driven by beta- and alpha-adrenergic receptor signaling, respectively. Clinically utilized adrenergic receptor antagonists can prevent cardiac injury and normalize vascular function. We found that tumor necrosis factor (TNF) gene deletion prevents the augmentation of myogenic reactivity in SAH: since membrane-bound TNF serves as a mechanosensor in the arteries assessed, alpha-adrenergic signaling putatively augments myogenic vasoconstriction by enhancing mechanosensor activity.