Search

Natural killer (NK) cells play a pivotal role in the immune response against viral infections, including SARS-CoV-2. However, our understanding of memory NK cell responses in the context of SARS-CoV-2 remains limited. To address this, we investigated the memory-like response of NK cells to SARS-CoV-2 peptides, presented by autologous cells. Blood samples from 45 donors underwent analysis for SARS-CoV-2 IgG antibodies, categorizing them into four groups based on the antibody kind and level. NK cells from SARS-CoV-2-experienced donors demonstrated enhanced degranulation and activation levels, IFNγ production and proliferative potential in response to SARS-CoV-2 peptides. Investigation of highly proliferating NK cells demonstrated the formation of distinct clusters depending on the SARS-CoV-2 peptide supplementation and the donor group. RNA sequencing revealed differential gene expression patterns, highlighting metabolism, protein transport, and immune response genes. Notably, KIR2DS4 expression correlated with enhanced IFNγ production, degranulation and proliferation levels, suggesting a role in SARS-CoV-2 recognition. Collectively, these findings provide detailed insights into antigen-specific NK cell responses to SARS-CoV-2 peptides, indicating potential mechanisms underlying NK cell activation in antiviral immunity., (© 2024 Wiley Periodicals LLC.)

Lymphotoxin α and lymphotoxin β (LTs), TNF superfamily members, are expressed in either soluble (LTα 3 ) or membrane-bound (LTα 1 β 2 or LTα 2 β 1 ) forms. In the pathological context, LT-mediated signaling is known to exacerbate autoimmunity by perpetuating inflammation and promoting the formation of tertiary lymphoid organs. Despite this understanding, the exact roles of LTα and LTβ in the pathogenesis of the murine model of multiple sclerosis, and experimental autoimmune encephalomyelitis (EAE), remain controversial. Here, we employed a panel of gene-modified mice with cell-type restricted ablation of LTα (targeting both membrane-bound and soluble forms of LTs) to unravel the contributions of LTs from various lymphoid cells, namely T cells, type 3 innate lymphoid cells (ILC3) and B cells, in EAE. We found that the effects of LTα deletion were dependent on the cellular source. ILC3-derived lymphotoxins exerted a protective role in EAE by regulating the accumulation of IFN-ɣ- and GM-CSF-producing T H cells in the CNS. In contrast, T-cell-derived lymphotoxins promoted IL-17A- and GM-CSF-mediated T H responses in the periphery, whereas B-cell-derived lymphotoxins were pathogenic only in the autoantibody-mediated EAE model. Collectively, our findings unveil the multifaceted involvement of lymphotoxins in EAE pathogenesis and challenge the view that lymphotoxins play a solely pathogenic role in neuroinflammation., (© 2024 Wiley‐VCH GmbH.)

Tumor necrosis factor (TNF) is one of many cytokines - protein molecules responsible for communication between the cells of immune system. TNF was discovered and given its grand name because of its striking antitumor effects in experimental systems, but its main physiological functions in the context of whole organism turned out to be completely unrelated to protection against tumors. This short review discusses "man-made" mouse models generated by early genome-editing technologies, which enabled us to establish true functions of TNF in health and certain diseases as well as to unravel potential strategies for improving therapy of TNF-dependent diseases.

In recent years, the role of cellular metabolism in immunity has come into the focus of many studies. These processes form a basis for the maintenance of tissue integrity and homeostasis, as well as represent an integral part of the immune response, in particular, inflammation. Metabolic adaptations not only ensure energy supply for immune response, but also affect the functions of immune cells by controlling transcriptional and post-transcriptional programs. Studying the immune cell metabolism facilitates the search for new treatment approaches, especially for metabolic disorders. Macrophages, innate immune cells, are characterized by a high functional plasticity and play a key role in homeostasis and inflammation. Depending on the phenotype and origin, they can either perform various regulatory functions or promote inflammation state, thus exacerbating the pathological condition. Furthermore, their adaptations to the tissue-specific microenvironment influence the intensity and type of immune response. The review examines the effect of metabolic reprogramming in macrophages on the functional activity of these cells and their polarization. The role of immunometabolic adaptations of myeloid cells in tissue homeostasis and in various pathological processes in the context of inflammatory and metabolic diseases is specifically discussed. Finally, modulation of the macrophage metabolism-related mechanisms reviewed as a potential therapeutic approach.

Multiple sclerosis (MS) is a complex autoimmune disease of central nervous system (CNS) characterized by the myelin sheath destruction and compromised nerve signal transmission. Understanding molecular mechanisms driving MS development is critical due to its early onset, chronic course, and therapeutic approaches based only on symptomatic treatment. Cytokines are known to play a pivotal role in the MS pathogenesis with interleukin-6 (IL-6) being one of the key mediators. This study investigates contribution of IL-6 produced by microglia and dendritic cells to the development of experimental autoimmune encephalomyelitis (EAE), a widely used mouse model of MS. Mice with conditional inactivation of IL-6 in the CX 3 CR1 + cells, including microglia, or CD11c + dendritic cells, displayed less severe symptoms as compared to their wild-type counterparts. Mice with microglial IL-6 deletion exhibited an elevated proportion of regulatory T cells and reduced percentage of pathogenic IFNγ-producing CD4+ T cells, accompanied by the decrease in pro-inflammatory monocytes in the CNS at the peak of EAE. At the same time, deletion of IL-6 from microglia resulted in the increase of CCR6 + T cells and GM-CSF-producing T cells. Conversely, mice with IL-6 deficiency in the dendritic cells showed not only the previously described increase in the proportion of regulatory T cells and decrease in the proportion of T H 17 cells, but also reduction in the production of GM-CSF and IFNγ in the secondary lymphoid organs. In summary, IL-6 functions during EAE depend on both the source and localization of immune response: the microglial IL-6 exerts both pathogenic and protective functions specifically in the CNS, whereas the dendritic cell-derived IL-6, in addition to being critically involved in the balance of regulatory T cells and T H 17 cells, may stimulate production of cytokines associated with pathogenic functions of T cells.

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., (© 2023 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Gubernatorova, Gorshkova, Bondareva, Podosokorskaya, Sheynova, Yakovleva, Bonch-Osmolovskaya, Nedospasov, Kruglov and Drutskaya.)

A highly effective humoral immune response induced by the Sputnik V vaccine was demonstrated in independent studies, as well as in large-scale post-vaccination follow-up studies. However, the shifts in the cell-mediated immunity induced by Sputnik V vaccination are still under investigation. This study was aimed at estimating the impact of Sputnik V on activating and inhibitory receptors, activation and proliferative senescence markers in NK and T lymphocytes. The effects of Sputnik V were evaluated by the comparison of PBMC samples prior to vaccination, and then three days and three weeks following the second (boost) dose. The prime-boost format of Sputnik V vaccination induced a contraction in the T cell fraction of senescent CD57 + cells and a decrease in HLA-DR-expressing T cells. The proportion of NKG2A + T cells was down-regulated after vaccination, whereas the PD-1 level was not affected significantly. A temporal increase in activation levels of NK cells and NKT-like cells was recorded, dependent on whether the individuals had COVID-19 prior to vaccination. A short-term elevation of the activating NKG2D and CD16 was observed in NK cells. Overall, the findings of the study are in favor of the Sputnik V vaccine not provoking a dramatic phenotypic rearrangement in T and NK cells, although it induces their slight temporal non-specific activation.

The effectiveness of the antiviral immune response largely depends on the activation of cytotoxic T cells. The heterogeneous group of functionally active T cells expressing the CD56 molecule (NKT-like cells), that combines the properties of T lymphocytes and NK cells, is poorly studied in COVID-19. This work aimed to analyze the activation and differentiation of both circulating NKT-like cells and CD56 - T cells during COVID-19 among intensive care unit (ICU) patients, moderate severity (MS) patients, and convalescents. A decreased proportion of CD56 + T cells was found in ICU patients with fatal outcome. Severe COVID-19 was accompanied by a decrease in the proportion of CD8 + T cells, mainly due to the CD56 - cell death, and a redistribution of the NKT-like cell subset composition with a predominance of more differentiated cytotoxic CD8 + T cells. The differentiation process was accompanied by an increase in the proportions of KIR2DL2/3 + and NKp30 + cells in the CD56 + T cell subset of COVID-19 patients and convalescents. Decreased percentages of NKG2D + and NKG2A + cells and increased PD-1 and HLA-DR expression levels were found in both CD56 - and CD56 + T cells, and can be considered as indicators of COVID-19 progression. In the CD56 - T cell fraction, increased CD16 levels were observed in MS patients and in ICU patients with lethal outcome, suggesting a negative role for CD56 - CD16 + T cells in COVID-19. Overall, our findings suggest an antiviral role of CD56 + T cells in COVID-19.

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.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Gorshkova, Gubernatorova, Dvorianinova, Yurakova, Marey, Averina, Holtze, Hildebrandt, Dmitriev, Drutskaya, Vyssokikh and Nedospasov.)

Coronavirus disease 2019 (COVID-19), caused by the SARS-CoV-2 virus, is accompanied by a dysregulated immune response. In particular, NK cells, involved in the antiviral response, are affected by the infection. This study aimed to investigate circulating NK cells with a focus on their activation, depletion, changes in the surface expression of key receptors, and functional activity during COVID-19, among intensive care unit (ICU) patients, moderately ill patients, and convalescents (CCP). Our data confirmed that NK cell activation in patients with COVID-19 is accompanied by changes in circulating cytokines. The progression of COVID-19 was associated with a coordinated decrease in the proportion of NKG2D + and CD16 + NK cells, and an increase in PD-1, which indicated their exhaustion. A higher content of NKG2D + NK cells distinguished surviving patients from non-survivors in the ICU group. NK cell exhaustion in ICU patients was additionally confirmed by a strong negative correlation of PD-1 and natural cytotoxicity levels. In moderately ill patients and convalescents, correlations were found between the levels of CD57, NKG2C, and NKp30, which may indicate the formation of adaptive NK cells. A reduced NKp30 level was observed in patients with a lethal outcome. Altogether, the phenotypic changes in circulating NK cells of COVID-19 patients suggest that the intense activation of NK cells during SARS-CoV-2 infection, most likely induced by cytokines, is accompanied by NK cell exhaustion, the extent of which may be critical for the disease outcome.

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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.

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, PGE 2 , 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-PGE 2 /PGE 2 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., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

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.

SARS-CoV-2 is a new coronavirus that is the cause of COVID-19 pandemic. To enter the cell, the virus interacts via its surface S protein with angiotensin-converting enzyme 2 (ACE2), the main entry receptor on the cell membrane. Most of protective antibodies, including those induced by vaccinations, target the S protein, preventing its interaction with the ACE2 receptor. We have evaluated an alternative strategy for blocking the S-ACE2 interaction using new antipeptide antibodies to the N-terminus of the ACE2 molecule. These antibodies allow detection of human ACE2 in vitro and ex vivo., (© 2022. Pleiades Publishing, Ltd.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Gubernatorova, Namakanova, Gorshkova, Medvedovskaya, Nedospasov and Drutskaya.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Gogoleva, Atretkhany, Dygay, Yurakova, Drutskaya and Nedospasov.)

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 TNFβ, 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.

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., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Authors.)

Systemic cytokine inhibition may be an effective therapeutic strategy for several autoimmune diseases. However, recent studies suggest that tissue or cell type-specific targeting of certain cytokines, including TNF, may have distinct advantages and show fewer side effects. Here we describe protocols for generating and testing bispecific cytokine inhibitors using variable domain of single-chain antibodies from Camelidae (V H H) with a focus on cell-specific TNF inhibitors.

Spatial organization and conformational changes of antibodies may significantly affect their biological functions. We assessed the effect of mutual organization of the two V H 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 V H 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., (© 2020 Federation of European Biochemical Societies.)

Systemic TNF neutralization can be used as a therapy for several autoimmune diseases. To evaluate the effects of cell type-restricted TNF blockade, we previously generated bispecific antibodies that can limit TNF secretion by myeloid cells (myeloid cell-specific TNF inhibitors or MYSTIs). In this study several such variable domain (VH) of a camelid heavy-chain only antibody-based TNF inhibitors were compared in relevant experimental models, both in vitro and in vivo. Pretreatment with MYSTI-2, containing the anti-F4/80 module, can restrict the release of human TNF (hTNF) from LPS-activated bone marrow-derived macrophage (BMDM) cultures of humanized TNF knock-in (mice; hTNFKI) more effectively than MYSTI-3, containing the anti-CD11b module. MYSTI-2 was also superior to MYSTI-3 in providing in vivo protection in acute toxicity model. Finally, MYSTI-2 was at least as effective as Infliximab in preventing collagen antibody-induced arthritis. This study demonstrates that a 33 kDa bispecific mini-antibody that specifically restricts TNF secretion by macrophages is efficient for amelioration of experimental arthritis., (©2020 Society for Leukocyte Biology.)

TNF is a key proinflammatory and immunoregulatory cytokine whose deregulation is associated with the development of autoimmune diseases and other pathologies. Recent studies suggest that distinct functions of TNF may be associated with differential engagement of its two receptors: TNFR1 or TNFR2. In this review, we discuss the relative contributions of these receptors to pathogenesis of several diseases, with the focus on autoimmunity and neuroinflammation. In particular, we discuss the role of TNFRs in the development of regulatory T cells during neuroinflammation and recent findings concerning targeting TNFR2 with agonistic and antagonistic reagents in various murine models of autoimmune and neuroinflammatory disorders and cancer., (©2020 Society for Leukocyte Biology.)

COVID-19 mortality is strongly associated with the development of severe pneumonia and acute respiratory distress syndrome with the worst outcome resulting in cytokine release syndrome and multiorgan failure. It is becoming critically important to identify at the early stage of the infection those patients who are prone to develop the most adverse effects. Elevated systemic interleukin-6 levels in patients with COVID-19 are considered as a relevant parameter in predicting most severe course of disease and the need for intensive care. This review discusses the mechanisms by which IL-6 may possibly contribute to disease exacerbation and the potential of therapeutic approaches based on anti-IL-6 biologics., Competing Interests: Declaration of Competing Interest The authors declare that there is no conflict of interest., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

In spite of successful therapeutic neutralization of proinflammatory cytokines in several autoimmune diseases, such therapy is not entirely free of side effects. The main reason relates to the fact that cytokine signaling may have protective components that need to be spared. Several approaches to achieve a less damaging cytokine inhibition are being explored. In our experimental studies we are using bispecific reagents based on VHH-modules from the heavy-chain-only antibodies to limit bioavailability of TNF and IL-6 produced by myeloid cells. After evaluation of their properties in vitro and in vivo we argue that these types of reagents may have an advantage over systemic blockers., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Dysregulated proinflammatory cytokine expression may result in the development of severe pathologies, such as rheumatoid arthritis, psoriasis, and neurodegenerative diseases. Transgenic mice and, in particular, those with controllable systemic overexpression of proinflammatory cytokines have recently become an essential instrument to study the molecular mechanisms underlying disease development. Importantly, many of the models are humanized by introducing a human cytokine gene, while leaving or removing the respective endogenous mouse gene. Humanized mice are especially valuable for biomedical research as they provide a relevant model to develop therapies based on blocking the pathogenic activity of a cytokine or to establish the functional significance of genome polymorphisms. The review discusses the available humanized mouse models with overexpression of key proinflammatory cytokines (TNF, IL-ip, and IL-6) and inflammatory cytokines with more specific functions (IL-8, IL-17, and IL-32) and their significance for basic and clinical research.

Recently, much attention has been drawn to unraveling the mechanisms of neurodegenerative and neuroinflammatory disease pathogenesis. A special role in the development of neuropathologies is assigned to the interaction of the nervous and the immune systems. Microglia are the cells of the immune system that function as resident macrophages of the central nervous system (CNS) and are involved in the development of CNS, as well as in homeostatic interactions. Impaired microglia can contribute to neuroinflammation and neurodegeneration. With the help of genome editing technologies, the main paradigms in the development and functions of microglia have been addressed. At the same time, an understanding of the mechanisms of regulation of microglia in normal and pathological conditions is necessary to create an effective therapy aimed at treating various neurological diseases. This review focuses on recent findings on the origin of microglia, its regulatory role in the central nervous system, as well as its contribution to the development of neuroinflammation.

Skin wound healing is subject to an intricate regulation, involves many cell populations and molecular mediators, and is one of the key mechanisms that ensures the barrier functions of the skin and the maintenance of body homeostasis. The efficiency of this process is largely determined by the balance of proinflammatory and proregenerative signals, which are mediated by cytokines. The review summarizes the latest data on the role of proinflammatory cytokines, mainly tumor necrosis factor (TNF), interleukin 6 (IL-6), interleukin 1 (IL-1), and interferons (IFNs), in skin wound healing, including those obtained with the use of genome editing techniques and methods of reverse genetics to establish relevant animal models. The roles that proinflammatory cytokines play at various stages of skin regeneration are discussed for both normal state and systemic pathologies, such as diabetes. Promising approaches to treating poorly healing wounds are summarized.

Severe asthma is a heterogeneous inflammatory disease of the airways, which requires treatment with high-dose inhaled corticosteroids or their systemic administration, yet often remains uncontrolled despite this therapy. Over the past decades, research efforts into phenotyping of severe asthma and defining the pathological mechanisms of this disease were successful largely due to the development of appropriate animal models. Recent identification of distinct inflammatory patterns of severe asthma endotypes led to novel treatment approaches, including targeting specific cytokines or their receptors with neutralizing antibodies. Here we discuss how different experimental mouse models contributed to generation of clinically relevant findings concerning pathogenesis of severe asthma and to identification of potential targets for biologic therapy., (Copyright © 2019 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.)

TNF is a multifunctional cytokine involved in autoimmune disease pathogenesis that exerts its effects through two distinct TNF receptors, TNFR1 and TNFR2. While TNF- and TNFR1-deficient (but not TNFR2-deficient) mice show very similar phenotypes, the significance of TNFR2 signaling in health and disease remains incompletely understood. Recent studies implicated the importance of the TNF/TNFR2 axis in T regulatory (T reg ) cell functions. To definitively ascertain the significance of TNFR2 signaling, we generated and validated doubly humanized TNF/TNFR2 mice, with the option of conditional inactivation of TNFR2. These mice carry a functional human TNF-TNFR2 (hTNF-hTNFR2) signaling module and provide a useful tool for comparative evaluation of TNF-directed biologics. Conditional inactivation of TNFR2 in FoxP3 + cells in doubly humanized TNF/TNFR2 mice down-regulated the expression of T reg signature molecules (such as FoxP3, CD25, CTLA-4, and GITR) and diminished T reg suppressive function in vitro. Consequently, T reg -restricted TNFR2 deficiency led to significant exacerbation of experimental autoimmune encephalomyelitis (EAE), accompanied by reduced capacity to control Th17-mediated immune responses. Our findings expose the intrinsic and beneficial effects of TNFR2 signaling in T reg cells that could translate into protective functions in vivo, including treatment of autoimmunity., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Despite decades of research, the goal of achieving scarless wound healing remains elusive. One of the approaches, treatment with polymeric microcarriers, was shown to promote tissue regeneration in various in vitro models of wound healing. The in vivo effects of such an approach are attributed to transferred cells with polymeric microparticles functioning merely as inert scaffolds. We aimed to establish a bioactive biopolymer carrier that would promote would healing and inhibit scar formation in the murine model of deep skin wounds. Here we characterize two candidate types of microparticles based on fibroin/gelatin or spidroin and show that both types increase re-epithelialization rate and inhibit scar formation during skin wound healing. Interestingly, the effects of these microparticles on inflammatory gene expression and cytokine production by macrophages, fibroblasts, and keratinocytes are distinct. Both types of microparticles, as well as their soluble derivatives, fibroin and spidroin, significantly reduced the expression of profibrotic factors Fgf2 and Ctgf in mouse embryonic fibroblasts. However, only fibroin/gelatin microparticles induced transient inflammatory gene expression and cytokine production leading to an influx of inflammatory Ly6C+ myeloid cells to the injection site. The ability of microparticle carriers of equal proregenerative potential to induce inflammatory response may allow their subsequent adaptation to treatment of wounds with different bioburden and fibrotic content.

Asthma is a common inflammatory disease of the airway caused by a combination of genetic and environmental factors and characterized by airflow obstruction, wheezing, eosinophilia, and neutrophilia of lungs and sputum. Similar to other proinflammatory cytokines, IL-6 is elevated in asthma and plays an active role in this disease. However, the exact molecular mechanism of IL-6 involvement in the pathogenesis of asthma remains largely unknown and the major cellular source of pathogenic IL-6 has not been defined. In the present study, we used conditional gene targeting to demonstrate that macrophages and dendritic cells are the critical sources of pathogenic IL-6 in acute HDM-induced asthma in mice. Complete genetic inactivation of IL-6 ameliorated the disease with significant decrease in eosinophilia in the lungs. Specific ablation of IL-6 in macrophages reduced key indicators of type 2 allergic inflammation, including eosinophil and Th2 cell accumulation in the lungs, production of IgE and expression of asthma-associated inflammatory mediators. In contrast, mice with deficiency of IL-6 in dendritic cells demonstrated attenuated neutrophilic, but regular eosinophilic response in HDM-induced asthma. Taken together, our results indicate that IL-6 plays a pathogenic role in the HDM-induced asthma model and that lung macrophages and dendritic cells are the predominant sources of pathogenic IL-6 but contribute differently to the disease.

Reverse genetics approach, involving genome editing, makes it possible not only to establish the nonredundant and unique functions of genes and their products, but also to construct animal models for biomedical research. Interleukin 6 (IL-6) is an important immunoregulatory and proinflammatory cytokine that differs from many related proteins in having a rather complicated signal transduction scheme. Apart from the multiple functions of IL-6, the most relevant biological problem of recent years was establishing what cells produce IL-6, in what form IL-6 is produced, what cells are recipients of the IL-6 signal, and what are the downstream events and physiological consequences of the IL-6 signaling cascade. Because IL-6 is involved in the pathogenesis of many diseases and is a drug target, understanding the mechanisms of its normal and pathogenic effects is important for the clinics. The review summarizes the recent data available in the field.

To model human interleukin-6 (hIL-6) associated diseases, unique mice with transgenic overexpression of human IL-6 and reporter fluorescent protein EGFP in cells of macrophage-monocyte lineage were generated using loxP-Cre system. High level of hIL-6 production by macrophages and monocytes, as confirmed in vitro in primary culture of bone marrow-derived macrophages, in vivo resulted in early postnatal death in vivo, presumably, due to the effect of overexpression of hIL-6 on hematopoiesis.

Cytokines play a pivotal role in maintaining homeostasis of the immune system and in regulation of the immune response. Cytokine dysregulation is often associated with development of various pathological conditions, including autoimmunity. Recent studies have provided insights into the cytokine signaling pathways that are involved not only in pathogenesis of autoimmune neuroinflammatory disorders, such as multiple sclerosis, but also in neurodegenerative states, for example, Alzheimer's disease. Understanding the exact molecular mechanisms of disease pathogenesis and evaluation of relevant experimental animal models are necessary for development of effective therapeutic approaches.

Toll-like receptor 4 (TLR4) initiates immune response against Gram-negative bacteria upon specific recognition of lipid A moiety of lipopolysaccharide (LPS), the major component of their cell wall. Some natural differences between LPS variants in their ability to interact with TLR4 may lead to either insufficient activation that may not prevent bacterial growth, or excessive activation which may lead to septic shock. In this study we evaluated the biological activity of LPS isolated from pathogenic strain of Campylobacter jejuni , the most widespread bacterial cause of foodborne diarrhea in humans. With the help of hydrophobic chromatography and MALDI-TOF mass spectrometry we showed that LPS from a C. jejuni strain O2A consists of both hexaacyl and tetraacyl forms. Since such hypoacylation can result in a reduced immune response in humans, we assessed the activity of LPS from C. jejuni in mouse macrophages by measuring its capacity to activate TLR4-mediated proinflammatory cytokine and chemokine production, as well as NFκB-dependent reporter gene transcription. Our data support the hypothesis that LPS acylation correlates with its bioactivity.

Cytokines are involved in a wide range of functions shaping the normal immune response, yet inflammatory changes in the immune system due to dysregulated cytokine signaling may lead to the induction of autoimmunity. Cytokine inhibitors have revolutionized the treatment of many autoimmune diseases in recent years. Systemic cytokine ablation, however, is often associated with the development of adverse side effects and some patients simply do not respond to therapy. TNF, IL-1 and IL-6 are the best characterized proinflammatory cytokines considered as the main therapeutic targets for the treatment of several autoimmune and inflammatory diseases. But can anti-cytokine therapy become more selective and thus more efficient? This mini-review discusses several recently emerging paradigms and summarizes current experimental attempts to validate them in mouse studies., (Copyright © 2016. Published by Elsevier Ltd.)

Cytokine neutralization is successfully used for treatment of various autoimmune diseases and chronic inflammatory conditions. The complex biology of the two well-characterized proinflammatory cytokines TNF and IL-6 implicates unavoidable consequences when it comes to their global blockade. Because systemic cytokine ablation may result in unwanted side effects, efforts have been made to develop more specific cytokine inhibitors, which would spare the protective immunoregulatory functions of a given cytokine. In this article, we review current research and summarize new strategies for improved anti-TNF and anti-IL-6 biologics, which specifically target only selected parts of the signaling cascades mediated by these ligands., (© Society for Leukocyte Biology.)

Proinflammatory cytokines, such as TNF, IL-6, and IL-1, play pathogenic roles in multiple diseases and are attractive targets for biologic drugs. Because proinflammatory cytokines possess non-redundant protective and immunoregulatory functions, their systemic neutralization carries the potential for unwanted side effects. Therefore, next-generation anti-cytokine therapies would seek to selectively neutralize pathogenic cytokine signaling, leaving normal function intact. Fortunately, the biology of proinflammatory cytokines provides several such opportunities. Here, we discuss various applications of bispecific antibodies targeting cytokines with specific focus on selective TNF neutralization targeted directly to the surface of specific populations of monocytes and macrophages. These bispecific antibodies combine an anti-TNF V H H with V H Hs or scFvs directed against abundant surface molecules on myeloid cells and serve to limit the bioavailability of TNF produced by these cells. Such reagents may become prototypes of a novel class of anti-cytokine biologics.

Culturing of allogeneic or autologous cells in three-dimensional bioresorbable scaffolds is an important step in the engineering of constructs for regenerative medicine, as well as for experimental systems to study the mechanisms of cell differentiation and cell-to-cell interaction. Artificial substrates can modulate the phenotype and functional activity of immobilized cells. Investigating these changes is important for understanding the fundamental processes underlying cellular interactions in a 3D microenvironment and for improving tissue-engineered structures. In this study, we investigated the expression of the ICAM-1 adhesion molecule in mouse embryonic fibroblasts (MEF) when cultured on gelatin-fibroin scaffolds. Increased expression of ICAM-1 in MEF was detected only under 3D culture conditions both at the mRNA and protein levels. At the same time, the MEF cultured on various substrates did not oerexpress MAdCAM-1, indicating the selective effect of 3D culture conditions on ICAM-1 expression. One possible mechanism for ICAM-1 induction in MEF is associated with the activation of AP-1, since expression of c-Fos and Junb (but not cJun and Jund) was increased in MEF in 3D. When cultured under 2D conditions, the expression level of AP-1 components did not change.

Skin stem cells can regenerate epidermal appendages; however, hair follicles (HF) lost as a result of injury are barely regenerated. Here we show that macrophages in wounds activate HF stem cells, leading to telogen-anagen transition (TAT) around the wound and de novo HF regeneration, mostly through TNF signalling. Both TNF knockout and overexpression attenuate HF neogenesis in wounds, suggesting dose-dependent induction of HF neogenesis by TNF, which is consistent with TNF-induced AKT signalling in epidermal stem cells in vitro. TNF-induced β-catenin accumulation is dependent on AKT but not Wnt signalling. Inhibition of PI3K/AKT blocks depilation-induced HF TAT. Notably, Pten loss in Lgr5 + HF stem cells results in HF TAT independent of injury and promotes HF neogenesis after wounding. Thus, our results suggest that macrophage-TNF-induced AKT/β-catenin signalling in Lgr5 + HF stem cells has a crucial role in promoting HF cycling and neogenesis after wounding.

The link between inflammation and cancer was first proposed by R. Virchow. It was later realized that it is chronic inflammation that may promote cancer, whereas acute inflammation can actually block tumor development or even result in cure. Many molecular mediators of these diverse processes have been characterized only during the past 3 decades thanks to the advances in molecular and cellular techniques, as well as due to technologies of reverse genetics. In this chapter we discuss the role of Toll-like receptor (TLR) 4 signaling in cancer and contributions of proinflammatory cytokine signaling (whose expression may be driven by TLR-mediated signals) to tumor-promoting microenvironment. We also discuss recent clinical advances to target these pro-tumorigenic pathways at distinct stages of tumorigenesis., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Relationship between inflammation and cancer is now well-established and represents a paradigm that our immune response does not necessarily serves solely to protect us from infections and cancer. Many specific mechanisms that link chronic inflammation to cancer promotion and metastasis have been uncovered in the recent years. Here we are focusing on the effects that tumors may exert on inflammatory cascades, tuning the immune system ability to cause tumor promotion or regression. In particular, we discuss the contributions of chemokines, cytokines and exosomes to the processes such as induction of inflammation and tumorigenesis. Overall, tumor-elicited inflammation is a key driver of tumor progression and an essential component of tumor microenvironment., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The combined effect of innate immunity receptors in viral-bacterial coinfections was studied in vitro using the primary culture of murine macrophages activated by different combinations of ligands of innate immunity receptors belonging to the family of Toll-like receptors. The activation of macrophages first with a viral ligand and then with a bacterial one significantly decreased the expression of proinflammatory cytokine genes. Such attenuation of immune responses may occur during the development of bacterial complications in viral infections.

Myeloid-derived suppressor cells represent a heterogeneous population of immature myeloid cells. Under normal conditions, these cells differentiate into macrophages, dendritic cells, and granulocytes. However, in pathological states such as inflammation, infection, or tumor growth, there is an arrest of their differentiation that results in the accumulation of immature myeloid cells in the organism. In addition, these cells acquire a suppressor phenotype, expressing anti-inflammatory cytokines and reactive oxygen and nitrogen species, and suppress T-cell immune response. Myeloid-derived suppressor cells (MDSC) contribute to cancerogenesis by forming a favorable microenvironment for tumor growth. Proinflammatory cytokines, secreted by tumor cells and the tumor microenvironment, induce angiogenesis and metastasis and promote tumor growth. They also provide signals necessary for survival, accumulation, and function of MDSC. Understanding the mechanisms of myeloid suppressor cell development and the use of proinflammatory cytokine inhibitors may prove beneficial for tumor therapy.

Acute diseases of the respiratory tract are often caused by viral pathogens and accompanying secondary bacterial infections. It is known that the development of such bacterial complications is caused mainly by a decreased infiltration with immune system cells and by suppressed inflammation in the lungs. There are significant advances in understanding the mechanisms of secondary infections, although many details remain unclear. This review summarizes current knowledge of the molecular and cellular changes in the host organism that can influence the course of bacterial coinfections in the respiratory tract.

Tumor necrosis factor (TNF) is a pleiotropic cytokine that regulates many important processes in the body. TNF production in a physiological state supports the structure of lymphoid organs and determines the development of lymphoid cells in hematopoiesis. However, chronic TNF overexpression leads to the development of various autoimmune disorders. Sites of TNF production in the naïve state remain unclear due to the lack of in vivo models. In the present study, we used TNF-2A-Kat reporter mice to monitor the expression of TNF in different tissues. Comparative analysis of tissue fluorescence in TNF-2A-Kat reporter mice and wild type mice revealed constitutive expression of TNF in the skin of naïve adult mice. In the skin of TNF-2A-Kat reporter mouse embryos, no statistically significant differences in the expression of TNF compared to wild type animals were observed. Furthermore, we established that local depletion of microflora with topical antibiotics leads to a reduction in the fluorescence signal. Thus, we assume that the skin microflora is responsible for the expression of TNF in the skin of mice.

The process of tissue regeneration following damage takes place with direct participation of the immune system. The use of biomaterials as scaffolds to facilitate healing of skin wounds is a new and interesting area of regenerative medicine and biomedical research. In many ways, the regenerative potential of biological material is related to its ability to modulate the inflammatory response. At the same time, all foreign materials, once implanted into a living tissue, to varying degree cause an immune reaction. The modern approach to the development of bioengineered structures for applications in regenerative medicine should be directed toward using the properties of the inflammatory response that improve healing, but do not lead to negative chronic manifestations. In this work, we studied the effect of microcarriers comprised of either fibroin or fibroin supplemented with gelatin on the dynamics of the healing, as well as inflammation, during regeneration of deep skin wounds in mice. We found that subcutaneous administration of microcarriers to the wound area resulted in uniform contraction of the wounds in mice in our experimental model, and microcarrier particles induced the infiltration of immune cells. This was associated with increased expression of proinflammatory cytokines TNF, IL-6, IL-1β, and chemokines CXCL1 and CXCL2, which contributed to full functional recovery of the injured area and the absence of fibrosis as compared to the control group.