Alveolar macrophages (AMs) comprise the predominant immune cell population in the lungs, maintaining homeostasis and providing the first line of immune defense against various respiratory diseases. Most studies focus on macrophages differentiated from bone marrow precursors in vitro. However, the ontogeny of the tissue-resident macrophages and the lung microenvironment significantly determine their properties and functions, fundamentally distinguishing AMs from the cells derived in vitro. The use of AMs ex vivo, which maximally preserves their original phenotype and proliferative potential, is the most physiological and informative approach for studying various aspects of innate immune responses in lung diseases. Improving methods for their isolation and culturing remains an important task for obtaining relevant data on the functions of these cells. In this study, we evaluated the effect of enzymatic treatment, the most common method for detaching cultured AMs, on their phenotype. It was found that enzymatic treatment led to the decrease in production of GM-CSF and VEGF by the AMs mediators of myeloid cell differentiation and maturation, and angiogenesis, respectively, both in the non-activated state and in response to one of the most common allergens – house dust mite extract (HDM). Enzymatic treatment promoted the formation of a pro-inflammatory phenotype, manifested by the increased production of IL-6, as well as chemokines CCL4 and CCL5, which attract monocytes and lymphocytes to the site of inflammation, along with the trend toward increased expression of the M1-associated genes such as Nos2 and Cd38, in response to HDM. Thus, enzymatic detachment of the tissue-resident AMs promotes M1 polarization, which predetermines a more pronounced response to the allergen in vitro. The presented data highlight disadvantages of the enzymatic treatment of AMs, increasing the risk of artifacts, and affecting reliability of the experimental results.

Allergic contact dermatitis (ACD) is a chronic inflammatory skin disorder the development of which is driven by allergen sensitization in peripheral lymphoid organs and local cutaneous inflammation. Lymphotoxin (LT) and its receptor LTβR are critical for lymphoid organogenesis and immune regulation in barrier tissues, but their role in ACD pathogenesis remains incompletely defined. This study aimed to delineate differential contribution of the LTβR-dependent signaling in oxazolone-induced dermatitis. We examined Lta knockout (Lta KO) mice, which lack both soluble LTα3 and membrane-bound isoforms LTα1β2/LTα2β1, and the Ltbr knockout (Ltbr KO) mice, both of which lack lymph nodes. ACD was induced by repeated oxazolone application to ear skin, with assessment of clinical severity, inflammation-associated gene expression, serum IgE levels, and immune cell composition in blood and spleen. Contrary to previous reports, the Lta KO mice developed dermatitis comparable to the wild-type (WT) mice, with elevated IgE production. In contrast, the Ltbr KO mice were substantially protected from the disease, exhibiting attenuated clinical inflammation, reduced ear swelling, and decreased Tslp expression in the lesional skin at the background of a lower proportion of circulating CD4+ T cells. These findings indicate that LTβR-dependent signaling is pathogenic in allergic skin inflammation, while LTα-mediated pathways are dispensable, suggesting a potential role for the other LTβR ligand, LIGHT, in ACD pathogenesis. Notably, ACD developed even in the absence of lymph nodes, highlighting the importance of local, skin-resident LTβR-dependent mechanisms in the disease development.

Elevated systemic production of interleukin-6 (IL-6) is associated with a broad spectrum of autoimmune and inflammatory diseases. Monoclonal antibody therapies targeting IL-6 or its receptor are widely used to neutralize the accompanying adverse effects. This study addresses the challenge of independently quantifying free and olokizumab (OKZ)-bound IL-6 in biological samples using a preclinical mouse model with regulated overexpression of human IL-6. We developed an enzyme-linked immunosorbent assay (ELISA), in which biotin-labeled OKZ served as a detecting agent, enabling in vivo tracking of dynamics of IL-6/OKZ immune complexes. OKZ effectively reduced free IL-6 levels in mice with cytokine overexpression for at least 7 days, while persisting in circulation as IL-6/OKZ complexes. Additionally, we assessed the kinetics of neutralizing anti-OKZ antibody formation in this preclinical model. The methods developed can be applied for clinical monitoring during anti-cytokine therapy and in preclinical studies using mouse models to evaluate the efficacy of such therapy in controlling IL-6-dependent inflammation in experimentally induced diseases.

Tumor necrosis factor (TNF) is rapidly induced after ischemic stroke, but its proposed cell-specific and sex-dependent functions during post-stroke inflammation remain insufficiently understood. Here, we investigated the role of microglia-derived TNF in the acute and subacute response to permanent middle cerebral artery occlusion (pMCAO). Tnf expression was transiently upregulated after stroke, becoming significant at 4 h, peaking at 12-24 h, and returning to baseline by 5 days. In situ hybridization confirmed strong Tnf expression in the infarct and peri-infarct regions. Whole-brain transcriptomic profiling showed that global TNF deficiency reshaped the early post-ischemic response, shifting it from microglia-associated phagocytic and wound-healing pathways toward an interferon-related inflammatory signature. To define the specific contribution of microglial TNF, we used inducible Cx3cr1CreER:Tnffl/fl mice. Microglial TNF deletion had no effect on infarct volume in males at 24 h or 5 days after pMCAO, but significantly increased infarct size in females at both time points. In both sexes, brain TNF levels peaked at 24 h and were significantly reduced in Cx3cr1CreER:Tnffl/fl mice, confirming microglia as a major source of early post-ischemic TNF. However, downstream consequences diverged by sex. At 5 days, male Cx3cr1CreER:Tnffl/fl mice showed reduced microglial reactivity and 18 kDa translocator protein (TSPO) signal, with no change in T-cell infiltration, and exhibited increased density of mature oligodendrocytes. In contrast, female Cx3cr1CreER:Tnffl/fl mice displayed enhanced microglial reactivity, increased TSPO binding, higher peri-infarct T-cell infiltration, and reduced oligodendrocyte density and myelin integrity. Together, these findings identify microglial TNF as a sex-dependent regulator of post-stroke inflammation and myelin injury.

Systemic blockade of proinflammatory cytokines such as IL-1, TNF, and IL-6 using therapeutic antibodies has proven effective in treating a wide range of autoimmune and other chronic inflammatory diseases. However, such blockade also suppresses non-redundant protective and homeostatic functions of cytokines, leading to a number of undesirable side effects. In this study, a novel bispecific mini-antibody featuring modules targeting human TNF and CD14 demonstrated efficacy in controlling TNF secretion from human peripheral blood monocytes. Administration of this antibody protected humanized TNF mice from lethal hepatotoxicity induced by a combination of LPS and D-galactosamine.

Escherichia coli is one of the most common producers of recombinant proteins, including therapeutic antibody fragments. However, the outer membrane of E. coli contains high levels of lipopolysaccharide (LPS, also known as endotoxin), which can activate innate immune receptors, trigger immune responses, and induce systemic inflammation that may progress to septic shock. Ensuring extremely low endotoxin levels in preparations intended for in vivo applications is critically important. In this study, we investigated the endotoxin content in preparations of the bispecific mini-antibody MYSTI-2 produced in two E. coli strains: the Rosetta strain, which synthesizes conventional LPS, and the ClearColi strain, which synthesizes potentially non-toxic form of LPS. Our results demonstrate that near-complete removal of LPS can be achieved only through the use of a non-ionic detergent during purification, regardless of the bacterial strain used for protein production.

The naked mole-rat (Heterocephalus glaber) is a long-lived rodent renowned for its remarkable resistance to cancer and age-related diseases. Its immune system has a unique cellular composition with a predominance of myeloid cells. Previously, we reported that naked mole-rat macrophages activated in vitro with lipopolysaccharide (LPS) and interferon-gamma produce significantly less nitric oxide (NO) compared to laboratory mouse (Mus musculus) macrophages. Furthermore, genes involved in arginine metabolism show distinct expression patterns between the two species following pro-inflammatory macrophage activation. To address the inflammation-induced species-specific profile of NO metabolism in vivo, a model of acute LPS-induced systemic inflammation was used. 24 h after LPS administration blood cell composition, intracellular NO production, expression of arginine metabolism-related genes and cytokines were analyzed in whole tissues as well as in sorted splenic CD11b + cells. LPS administration induced sickness behavior and increased inflammatory cytokine gene expression in the spleen and the liver of both naked mole-rats and mice. However, naked mole-rats exhibited a species-specific reduction in inducible NO-synthase (iNOS) activity: the intracellular NO levels were significantly elevated in myeloid blood cells after LPS administration in mice, but not in naked mole-rats. The expression of the gene coding for iNOS, Nos2, was upregulated in a dose-dependent manner in murine tissues, whereas in naked-mole rats, Nos2 induction was observed only after high-dose LPS administration in the liver. Additionally, an alternative arginine-utilizing pathway involved in creatine synthesis remained unaffected by LPS administration in naked mole-rat tissues, in contrast to mice. These findings suggest that naked mole-rats may tolerate acute inflammation due to metabolic adaptations that modulate iNOS activation in myeloid cells.

Endoplasmic reticulum (ER) stress and necroptosis are associated with the pathogenesis of inflammatory bowel disease (IBD); however, the potential crosstalk between these pathways is unclear. Here, we show that intestinal epithelial cell (IEC)-specific X-box binding protein 1 (XBP1) deficiency strongly aggravates the development of necroptosis-induced colitis, but not ileitis, in mice lacking caspase-8 or its adapter Fas associated with death domain (FADD) in IECs. Mechanistically, XBP1 ablation led to diminished mucin 2 (MUC2) expression and impaired mucus layer formation in the colon, which allowed bacteria to penetrate and reach the epithelial surface. This was not sufficient to trigger colitis in the presence of an intact epithelial monolayer but synergized with IEC necroptosis to induce severe colon inflammation. Our results revealed that XBP1 and caspase-8 control different components of the intestinal barrier that synergize to maintain mucosal immune homeostasis and prevent colon inflammation. This could be relevant for the better understanding of the mechanisms causing IBD.

Anti-tumour necrosis factor (TNF) therapy has been successfully applied over the past 30 years to treat a number of autoimmune conditions. This research and development area has grown tremendously not only with regard to various clinical applications, but it also provided new insights into TNF immunobiology and allowed to critically evaluate several novel concepts of selective TNF inhibition.

The naked mole-rat (NMR) is a unique long-lived rodent with low cancer incidence. Understanding the molecular mechanisms that NMR evolved to control aging and tumorigenesis is important for biomedicine. It is commonly accepted that the immune system has essential functions in the tumor growth control in animals. In-depth study of the NMR immune system has recently begun, thus peculiarities of antitumor response in these animals remain undiscovered. However, it was shown that myelopoiesis predominates in NMR, therefore it can be assumed that the innate immune cells in the naked mole rat contribute to the successful control of the cancer incidence and tumor growth. This brief provides an overview of ongoing research into the properties of naked mole rat macrophages. Recent study shown that naked mole rat peritoneal macrophages are capable of acquiring an inflammatory phenotype (M1) but polarization into an anti-inflammatory phenotype (M2) under standard stimulus is limited. A more in-depth study using transcriptome sequencing and immunometabolic profiling in novel in vitro model of naked mole rat bone marrow macrophages suggested by our group revealed non-canonical features of M1 as well as M2 phenotypes of naked mole rat macrophages, which can be associated with the evolutionary adaptation of the species. Continued study of the different polarization conditions of naked mole rat macrophages is important to determine unique adaptations in NMR antitumor immunity.

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.

Key Points Proximal tubular TNF aggravates kidney injury and fibrogenesis in aristolochic acid nephropathy. Tubular TNF disrupts the cell cycle in injured tubular epithelial cells. TNF-mediated toxic renal injury is independent of systemic immune responses. Background Aristolochic acid nephropathy (AAN) presents with tubular epithelial cell (TEC) damage and tubulointerstitial inflammation. Although TNF- α regulates cell apoptosis and inflammatory responses, the effects of tubular TNF in the progression of AAN require elucidation. Methods Floxed TNF mice on the 129/SvEv background were crossed with PEPCK-Cre mice to generate PEPCK-Cre + TNF flox/flox (TNF PTKO) mice or bred with Ksp-Cre mice to generate KSP-Cre + TNF flox/flox (TNF DNKO) mice. TNF PTKO, TNF DNKO, and wild-type controls (Cre negative littermates) were subjected to acute and chronic AAN. Results Deletion of TNF in the proximal but not distal nephron attenuated kidney injury, renal inflammation, and tubulointerstitial fibrosis after acute or chronic aristolochic acid (AA) exposure. The TNF PTKO mice did not have altered numbers of infiltrating myeloid cells in AAN kidneys. Nevertheless, kidneys from AA-treated TNF PTKO mice had reduced levels of proteins involved in regulated cell death, higher proportions of TECs in the G0/G1 phase, and reduced TEC proportions in the G2/M phase. Pifithrin- α , which restores the cell cycle, abrogated differences between the wild-type and PTKO cohorts in G2/M phase arrest of TECs and kidney fibrosis after AA exposure. Conclusions TNF from the proximal but not the distal nephron propagates kidney injury and fibrogenesis in AAN in part by inducing G2/M cell cycle arrest of TECs.

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.

Chronic inflammation caused by overexpression of IL-6 underlies a number of pathological conditions Mouse models of systemic chronic inflammation with overexpression of human IL-6 (hIL-6) are in demand not only in the context of studying the molecular mechanisms of inflammation, but also in assessing the effectiveness of clinically approved or newly developed IL-6 inhibitors. One experimental approach in addressing such models in mice relies on the induction of systemic acute inflammation in response to lipopolysaccharide (LPS) administration. This work describes mice with tamoxifen-dependent overexpression of human IL-6 in CX3CR1+ myeloid cells in the context of systemic inflammation induced by LPS administration. Our study demonstrates that the highest expression of the transgene carrying IL6 was observed in the heart, while high production of this cytokine was detected in the blood serum. In response to LPS administration, the production of hIL-6 in the blood increased in transgenic mice, while the production of mIL-6 also increased and was comparable to that in wild-type mice. The consequences of high systemic production of hIL-6, which in our model originates from CX3CR1+ tissue-resident macrophages, were noticeable even in the organs in which these cells are not present. Thus, significant amounts of hIL-6 were detected in tissue lysates of the lungs of transgenic mice after LPS administration. Evaluation of the expression of genes encoding cytokines and markers of tissue remodeling upon injury using quantitative real-time PCR showed significant changes in their expression in the context of LPS-induced systemic inflammation. Thus, this work demonstrates the feasibility of using a mouse model with tamoxifen-dependent transgene activation in CX3CR1+ tissue-resident macrophages to study the effects of systemic overexpression of IL-6 and pharmacological blockade of this cytokine with clinically approved or newly developed inhibitors in the context of experimentally induced diseases.

Itaconate is one of the most studied immunometabolites produced by myeloid cells during inflammatory response. It mediates a wide range of anti-inflammatory and immunoregulatory effects and plays a role in a number of pathological states, including autoimmunity and cancer. Itaconate and its derivatives are considered potential therapeutic agents for the treatment of inflammatory diseases. While immunoregulatory effects of itaconate have been extensively studied in vitro and using knockout mouse models, less is known about how therapeutic administration of this metabolite regulates inflammatory response in vivo. Here, we investigate the immunoregulatory properties of exogenous administration of itaconate and its derivative dimethyl itaconate in a mouse model of lipopolysaccharide-induced inflammation. The data show that administration of itaconate or dimethyl itaconate controls systemic production of multiple cytokines, including increased IL-10 production. However, only dimethyl itaconate was able to suppress systemic production of IFN gamma and IL-1 beta. In contrast to in vitro data, administration of itaconate or dimethyl itaconate in vivo resulted in systemic upregulation of IL-6 in the blood. Electrophilic stress due to itaconate or dimethyl itaconate was not responsible for IL-6 upregulation. However, inhibition of succinate dehydrogenase with dimethyl malonate also resulted in elevated systemic levels of IL-6 and IL-10. Taken together, our study reports a novel effect of exogenous itaconate and its derivative dimethyl itaconate on the production of IL-6 in vivo, with important implications for the development of itaconate-based anti-inflammatory therapies. Itaconate and dimethyl itaconate shape cytokine response in vivo in a model of lipopolysaccharide-induced inflammation, including upregulation of IL-6 and IL-10. Graphical Abstract

This short review focuses on cytokines, molecular mediators of immunity and many other physiological functions of the body. Research on cytokines revealed new principles of cell-tocell communications and new paradigms in intracellular signal transduction. A few recombinant cytokines and, paradoxically, some cytokine inhibitors found practical use in medicine.

PDF - 395KB, Supplementary Figure 1: Structure of MO-TES391 and KIAA1864 transcripts.

PDF - 60KB, Supplementary Table 1: Serological reactivity of MO-TES391 assessed by SMARTA.