Basophil-derived tumor necrosis factor can enhance survival in a sepsis model in mice.

Basophils are evolutionarily conserved in vertebrates, despite their small numbers and short life span, suggesting that they have beneficial roles in maintaining health. However, these roles are not fully defined. Here we demonstrate that basophil-deficient mice exhibit reduced bacterial clearance and increased morbidity and mortality in the cecal ligation and puncture (CLP) model of sepsis. Among the several proinflammatory mediators that we measured, tumor necrosis factor (TNF) was the only cytokine that was significantly reduced in basophil-deficient mice after CLP. In accordance with that observation, we found that mice with genetic ablation of Tnf in basophils exhibited reduced systemic concentrations of TNF during endotoxemia. Moreover, after CLP, mice whose basophils could not produce TNF, exhibited reduced neutrophil and macrophage TNF production and effector functions, reduced bacterial clearance, and increased mortality. Taken together, our results show that basophils can enhance the innate immune response to bacterial infection and help prevent sepsis.

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.

Stellate Cells, Hepatocytes, and Endothelial Cells Imprint the Kupffer Cell Identity on Monocytes Colonizing the Liver Macrophage Niche.

Macrophages are strongly adapted to their tissue of residence. Yet, little is known about the cell-cell interactions that imprint the tissue-specific identities of macrophages in their respective niches. Using conditional depletion of liver Kupffer cells, we traced the developmental stages of monocytes differentiating into Kupffer cells and mapped the cellular interactions imprinting the Kupffer cell identity. Kupffer cell loss induced tumor necrosis factor (TNF)- and interleukin-1 (IL-1) receptor-dependent activation of stellate cells and endothelial cells, resulting in the transient production of chemokines and adhesion molecules orchestrating monocyte engraftment. Engrafted circulating monocytes transmigrated into the perisinusoidal space and acquired the liver-associated transcription factors inhibitor of DNA 3 (ID3) and liver X receptor-α (LXR-α). Coordinated interactions with hepatocytes induced ID3 expression, whereas endothelial cells and stellate cells induced LXR-α via a synergistic NOTCH-BMP pathway. This study shows that the Kupffer cell niche is composed of stellate cells, hepatocytes, and endothelial cells that together imprint the liver-specific macrophage identity.

Inflammation-induced formation of fat-associated lymphoid clusters.

Fat-associated lymphoid clusters (FALCs) are a type of lymphoid tissue associated with visceral fat. Here we found that the distribution of FALCs was heterogeneous, with the pericardium containing large numbers of these clusters. FALCs contributed to the retention of B-1 cells in the peritoneal cavity through high expression of the chemokine CXCL13, and they supported B cell proliferation and germinal center differentiation during peritoneal immunological challenges. FALC formation was induced by inflammation, which triggered the recruitment of myeloid cells that expressed tumor-necrosis factor (TNF) necessary for signaling via the TNF receptors in stromal cells. Natural killer T cells (NKT cells) restricted by the antigen-presenting molecule CD1d were likewise required for the inducible formation of FALCs. Thus, FALCs supported and coordinated the activation of innate B cells and T cells during serosal immune responses.

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.

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.

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.

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.

[How T lymphocytes coordinate rheumatic inflammation]. / Wie T-Lymphozyten rheumatische Entzündung koordinieren.

Helper T (Th) cells play a decisive role in triggering and maintaining chronic rheumatic inflammation. Via secretion of proinflammatory cytokines and expression of costimulatory cell surface molecules, Th lymphocytes coordinate the recruitment and activation of effector cells, which are ultimately responsible for the immunopathology and tissue destruction. However, therapeutic approaches aimed at eliminating Th cells were unsuccessful due to their lack of selectivity. At the German Rheumatism Research Center (Deutsches Rheuma-Forschungszentrum, DRFZ), we are working to improve the understanding of the Th cells involved in chronic inflammatory reactions. Based on this understanding, our aim is to develop novel treatment strategies that selectively target the pathogenic Th lymphocytes causing rheumatic inflammation. The current article summarizes the DRFZ's research activities on this subject.

Myeloid tumor necrosis factor and heme oxygenase-1 regulate the progression of colorectal liver metastases during hepatic ischemia-reperfusion.

The liver ischemia-reperfusion (IR) injury that occurs consequently to hepatic resection performed in patients with metastases can lead to tumor relapse for not fully understood reasons. We assessed the effects of liver IR on tumor growth and the innate immune response in a mouse model of colorectal (CR) liver metastasis. Mice subjected to liver ischemia 2 days after intrasplenic injection of CR carcinoma cells displayed a higher metastatic load in the liver, correlating with Kupffer cells (KC) death through the activation of receptor-interating protein 3 kinase (RIPK3) and caspase-1 and a recruitment of monocytes. Interestingly, the immunoregulatory mediators, tumor necrosis factor-α (TNF-α) and heme oxygenase-1 (HO-1) were strongly upregulated in recruited monocytes and were also expressed in the surviving KC following IR. Using TNFflox/flox LysMcre/wt mice, we showed that TNF deficiency in macrophages and monocytes favors tumor progression after IR. The antitumor effect of myeloid cell-derived TNF involved direct tumor cell apoptosis and a reduced expression of immunosuppressive molecules such as transforming growth factor-ß, interleukin (IL)-10, inducible nitric oxyde synthase (iNOS), IL-33 and HO-1. Conversely, a monocyte/macrophage-specific deficiency in HO-1 (HO-1flox/flox LysMcre/wt ) or the blockade of HO-1 function led to the control of tumor progression post-liver IR. Importantly, host cell RIPK3 deficiency maintains the KC number upon IR, inhibits the IR-induced innate cell recruitment, increases the TNF level, decreases the HO-1 level and suppresses the tumor outgrowth. In conclusion, tumor recurrence in host undergoing liver IR is associated with the death of antitumoral KC and the recruitment of monocytes endowed with immunosuppressive properties. In both of which HO-1 inhibition would reinforce their antitumoral activity.

Intrinsic TNFR2 signaling in T regulatory cells provides protection in CNS autoimmunity.

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 (Treg) 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 Treg signature molecules (such as FoxP3, CD25, CTLA-4, and GITR) and diminished Treg suppressive function in vitro. Consequently, Treg-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 Treg cells that could translate into protective functions in vivo, including treatment of autoimmunity.

TNF-α in T lymphocytes attenuates renal injury and fibrosis during nephrotoxic nephritis.

Nephrotoxic serum nephritis (NTN) models immune-mediated human glomerulonephritis and culminates in kidney inflammation and fibrosis, a process regulated by T lymphocytes. TNF-α is a key proinflammatory cytokine that contributes to diverse forms of renal injury. Therefore, we posited that TNF-α from T lymphocytes may contribute to NTN pathogenesis. Here, mice with T cell-specific deletion of TNF-α (TNF TKO) and wild-type (WT) control mice were subjected to the NTN model. At 14 days after NTN, kidney injury and fibrosis were increased in kidneys from TNF TKO mice compared with WT mice. PD1+CD4+ T cell numbers and mRNA levels of IL-17A were elevated in NTN kidneys of TNF TKO mice, suggesting that augmented local T helper 17 lymphocyte responses in the TNF TKO kidney may exaggerate renal injury and fibrosis. In turn, we found increased accumulation of neutrophils in TNF TKO kidneys during NTN. We conclude that TNF-α production in T lymphocytes mitigates NTN-induced kidney injury and fibrosis by inhibiting renal T helper 17 lymphocyte responses and infiltration of neutrophils.

Contrasting contributions of TNF from distinct cellular sources in arthritis.

OBJECTIVES: Neutralisation of tumour necrosis factor (TNF) is widely used as a therapy for rheumatoid arthritis (RA). However, this therapy is only effective in less than a half of patients and is associated with several side effects. We hypothesised that TNF may possess non-redundant protective and immunomodulatory functions in vivo that cannot be blocked without a cost. The present work aimed to identify cellular sources of protective and pathogenic TNF, and its molecular forms during autoimmune arthritis. METHODS: Mice lacking TNF expression by distinct cell types, such as myeloid cells and T or B lymphocytes, were subjected to collagen-induced arthritis (CIA) and collagen antibody-induced arthritis. Mice lacking soluble TNF production were also employed. The severity and incidence of the disease, as well as humoral and cellular responses were assessed. RESULTS: Myeloid cell-derived TNF contributes to both induction and pathogenesis of autoimmune arthritis. Conversely, T cell-derived TNF is protective during the induction phase of arthritis via limiting of interleukin-12 production by dendritic cells and by subsequent control of autoreactive memory T cell development, but is dispensable during the effector phase of arthritis. B cell-derived TNF mediates severity of CIA via control of pathogenic autoantibody production. CONCLUSIONS: Distinct TNF-producing cell types may modulate disease development through different mechanisms, suggesting that in arthritis TNF ablation from restricted cellular sources, such as myeloid cells, while preserving protective TNF functions from other cell types may be superior to pan-anti-TNF therapy.

KLF4 in Macrophages Attenuates TNFα-Mediated Kidney Injury and Fibrosis.

BACKGROUND: Polarized macrophage populations can orchestrate both inflammation of the kidney and tissue repair during CKD. Proinflammatory M1 macrophages initiate kidney injury, but mechanisms through which persistent M1-dependent kidney damage culminates in fibrosis require elucidation. Krüppel-like factor 4 (KLF4), a zinc-finger transcription factor that suppresses inflammatory signals, is an essential regulator of macrophage polarization in adipose tissues, but the effect of myeloid KLF4 on CKD progression is unknown. METHODS: We used conditional mutant mice lacking KLF4 or TNFα (KLF4's downstream effector) selectively in myeloid cells to investigate macrophage KLF4's role in modulating CKD progression in two models of CKD that feature robust macrophage accumulation, nephrotoxic serum nephritis, and unilateral ureteral obstruction. RESULTS: In these murine CKD models, KLF4 deficiency in macrophages infiltrating the kidney augmented their M1 polarization and exacerbated glomerular matrix deposition and tubular epithelial damage. During the induced injury in these models, macrophage-specific KLF4 deletion also exacerbated kidney fibrosis, with increased levels of collagen 1 and α-smooth muscle actin in the injured kidney. CD11b+Ly6Chi myeloid cells isolated from injured kidneys expressed higher levels of TNFα mRNA versus wild-type controls. In turn, mice bearing macrophage-specific deletion of TNFα exhibited decreased glomerular and tubular damage and attenuated kidney fibrosis in the models. Moreover, treatment with the TNF receptor-1 inhibitor R-7050 during nephrotoxic serum nephritis reduced damage, fibrosis, and necroptosis in wild-type mice and mice with KLF4-deficient macrophages, and abrogated the differences between the two groups in these parameters. CONCLUSIONS: These data indicate that macrophage KLF4 ameliorates CKD by mitigating TNF-dependent injury and fibrosis.

Engulfment of mast cell secretory granules on skin inflammation boosts dendritic cell migration and priming efficiency.

BACKGROUND: Mast cells (MCs) are best known as key effector cells of allergic reactions, but they also play an important role in host defense against pathogens. Despite increasing evidence for a critical effect of MCs on adaptive immunity, the underlying mechanisms are poorly understood. OBJECTIVE: Here we monitored MC intercellular communication with dendritic cells (DCs), MC activation, and degranulation and tracked the fate of exocytosed mast cell granules (MCGs) during skin inflammation. METHODS: Using a strategy to stain intracellular MCGs in vivo, we tracked the MCG fate after skin inflammation-induced MC degranulation. Furthermore, exogenous MCGs were applied to MC-deficient mice by means of intradermal injection. MCG effects on DC functionality and adaptive immune responses in vivo were assessed by combining intravital multiphoton microscopy with flow cytometry and functional assays. RESULTS: We demonstrate that dermal DCs engulf the intact granules exocytosed by MCs on skin inflammation. Subsequently, the engulfed MCGs are actively shuttled to skin-draining lymph nodes and finally degraded inside DCs within the lymphoid tissue. Most importantly, MCG uptake promotes DC maturation and migration to skin-draining lymph nodes, partially through MC-derived TNF, and boosts their T-cell priming efficiency. Surprisingly, exogenous MCGs alone are sufficient to induce a prominent DC activation and T-cell response. CONCLUSION: Our study highlights a unique feature of peripheral MCs to affect lymphoid tissue-borne adaptive immunity over distance by modifying DC functionality through delivery of granule-stored mediators.

Commensal microbiota influence systemic autoimmune responses.

Antinuclear antibodies are a hallmark feature of generalized autoimmune diseases, including systemic lupus erythematosus and systemic sclerosis. However, the processes underlying the loss of tolerance against nuclear self-constituents remain largely unresolved. Using mice deficient in lymphotoxin and Hox11, we report that approximately 25% of mice lacking secondary lymphoid organs spontaneously develop specific antinuclear antibodies. Interestingly, we find this phenotype is not caused by a defect in central tolerance. Rather, cell-specific deletion and in vivo lymphotoxin blockade link these systemic autoimmune responses to the formation of gut-associated lymphoid tissue in the neonatal period of life. We further demonstrate antinuclear antibody production is influenced by the presence of commensal gut flora, in particular increased colonization with segmented filamentous bacteria, and IL-17 receptor signaling. Together, these data indicate that neonatal colonization of gut microbiota influences generalized autoimmunity in adult life.

Lymphotoxin beta receptor signaling in intestinal epithelial cells orchestrates innate immune responses against mucosal bacterial infection.

Epithelial cells provide the first line of defense against mucosal pathogens; however, their coordination with innate and adaptive immune cells is not well understood. Using mice with conditional gene deficiencies, we found that lymphotoxin (LT) from innate cells expressing transcription factor RORgammat, but not from adaptive T and B cells, was essential for the control of mucosal C. rodentium infection. We demonstrate that the LTbetaR signaling was required for the regulation of the early innate response against infection. Furthermore, we have revealed that LTbetaR signals in gut epithelial cells and hematopoietic-derived cells coordinate to protect the host from infection. We further determined that LTbetaR signaling in intestinal epithelial cells was required for recruitment of neutrophils to the infection site early during infection via production of CXCL1 and CXCL2 chemokines. These results support a model wherein LT from RORgammat(+) cells orchestrates the innate immune response against mucosal microbial infection.

Signaling via the MyD88 adaptor protein in B cells suppresses protective immunity during Salmonella typhimurium infection.

The myeloid differentiation primary response gene 88 (Myd88) is critical for protection against pathogens. However, we demonstrate here that MyD88 expression in B cells inhibits resistance of mice to Salmonella typhimurium infection. Selective deficiency of Myd88 in B cells improved control of bacterial replication and prolonged survival of the infected mice. The B cell-mediated suppressive pathway was even more striking after secondary challenge. Upon vaccination, mice lacking Myd88 in B cells became completely resistant against this otherwise lethal infection, whereas control mice were only partially protected. Analysis of immune defenses revealed that MyD88 signaling in B cells suppressed three crucial arms of protective immunity: neutrophils, natural killer cells, and inflammatory T cells. We further show that interleukin-10 is an essential mediator of these inhibitory functions of B cells. Collectively, our data identify a role for MyD88 and B cells in regulation of cellular mechanisms of protective immunity during infection.

Cell-type-restricted anti-cytokine therapy: TNF inhibition from one pathogenic source.

Overexpression of TNF contributes to pathogenesis of multiple autoimmune diseases, accounting for a remarkable success of anti-TNF therapy. TNF is produced by a variety of cell types, and it can play either a beneficial or a deleterious role. In particular, in autoimmunity pathogenic TNF may be derived from restricted cellular sources. In this study we evaluated the feasibility of cell-type-restricted TNF inhibition in vivo. To this end, we engineered MYSTI (Myeloid-Specific TNF Inhibitor)--a recombinant bispecific antibody that binds to the F4/80 surface molecule on myeloid cells and to human TNF (hTNF). In macrophage cultures derived from TNF humanized mice MYSTI could capture the secreted hTNF, limiting its bioavailability. Additionally, as evaluated in TNF humanized mice, MYSTI was superior to an otherwise analogous systemic TNF inhibitor in protecting mice from lethal LPS/D-Galactosamine-induced hepatotoxicity. Our results suggest a novel and more specific approach to inhibiting TNF in pathologies primarily driven by macrophage-derived TNF.