InVivoMAb anti-mouse IL-6R

Alcohol use disorder (AUD) represents a significant neurological health burden, yet the biological mechanisms underlying alcohol-induced brain pathology remain incompletely understood. Moreover, the molecular underpinnings of the transition from alcohol exposure to alcohol dependence are not well-characterized. We used mass spectrometry (MS)-based proteomics in a preliminary discovery study to compare cerebrospinal fluid (CSF) of alcohol-exposed Non-dependent (Non-dep) versus alcohol-dependent (Dep) mice that underwent the chronic intermittent ethanol (alcohol) - two-bottle choice (CIE-2 BCE) procedure and systemic anti-IL-6 Receptor antibody administration (n = 9; female = 5, male = 4). CSF samples from individual mice were processed for proteomic analysis and digested with trypsin overnight. Peptides were analyzed via data-independent acquisition (DIA)-MS and data were processed in DIA-NN at 1 % FDR. We identified 595 unique proteins across both groups, with 140 proteins differentially detected in CSF from Dep mice and 62 proteins specific to alcohol-exposed but Non-dep controls. The Dep-specific proteins revealed signatures of blood-brain barrier (BBB) disruption, neuroinflammation, cellular stress responses, and complement system activation. In contrast, Non-dep-specific proteins indicated preserved protective mechanisms including complement regulation, anti-inflammatory signaling, and neuronal calcium homeostasis. Ethanol-dependent-specific findings include MMP2, BIP, and to a lesser extent VE-cadherin (CDH5) and VCAM1, indicative of the beginnings of endothelial damage and BBB disruption, alongside established neuroinflammation markers GFAP, CHI3L1, and CX3CL1. This work provides novel preliminary protein-level evidence that alcohol exposure and alcohol dependence are dichotomous; despite the small sample size and limited power for moderate effect sizes, there appears to be a clear molecular transition from maintained protective mechanisms to vascular damage, BBB breakdown, and sustained neuroinflammation.

Sarcopenia, the age-related decline in muscle mass, strength and function, is characterized by impaired muscle homeostasis, reduced regenerative potential of muscle stem cells (MuSCs) and increased fibrosis. Here we report that aged MuSCs can autonomously instruct fibro-adipogenic progenitors (FAPs) to proliferate and acquire a fibrogenic phenotype, independent of other cell types. Both the polycomb-deficient Ezh2-/- mouse model and aged mice exhibited defective regeneration, FAP expansion, fibrosis and elevated secretion of interleukin 6 (IL-6) and secreted phosphoprotein 1 (Spp1; osteopontin) by MuSCs. In aged MuSCs, reduction of the histone H3K27me3 repressive mark at the Nfbk1 gene correlated with its increased expression and enhanced chromatin recruitment to the IL6 and Spp1 genes, leading to their activation. Pharmacological inhibition of IL-6 and Spp1 signaling in co-culture systems or in aged mice reduced FAP proliferation and muscle fibrosis. These findings indicate that epigenetic dysregulation of aged MuSCs contributes to aged-related muscle fibrosis.

Alcohol use disorder (AUD) is defined by the emergence of negative affective symptoms during withdrawal. Neuroinflammation is a key contributor to AUD, and it is well known to play an essential role in the pathogenesis of pain states. The chronic-intermittent ethanol two-bottle choice (CIE-2BC) paradigm is well-established to generate alcohol-dependent (Dep) and non-dependent (Non-Dep) mice. Our recent work demonstrated that the CIE-2BC model promotes mechanical allodynia in Dep mice, with these mice developing mechanical allodynia during withdrawal. In this study, we examined the role of interleukin-6 (IL-6) in the development of mechanical allodynia by adapting the CIE-2BC mouse model and employing the von Frey test, in situ hybridization (RNAscope), and Multiplex protein analysis of the spinal cord, examining changes in this target including an array of cytokines associated with IL-6 signaling. CIE-2BC escalated alcohol drinking and enhanced mechanical allodynia in Dep versus Non-Dep mice, with Dep females displaying greater alcohol intake. Dep mice displayed increased IL-6 protein in the spinal cord while males additionally had increased Il6+ cell expression versus Non-Dep controls. Systemic treatment of an IL-6 receptor antibody (IL-6R Ab) did not decrease mechanical allodynia during abstinence. Collectively, these data suggest that alcohol exerts sex-dependent effects on spinal IL-6 levels. However, in our study, blocking IL-6 signaling did not reduce alcohol-associated pain sensitivity in a mouse model of comorbid pain and alcohol dependence.

Preterm birth (PTB) is a major cause of neonatal mortality and morbidity. Evidence supports a determinant role for interleukin-6 (IL-6) in the pathophysiology of PTB. Our group developed a small peptide, HSJ633, that antagonizes the interleukin-6 receptor (IL-6R). Binding assays performed on HEK-Blue IL-6 cells reveal that HSJ633 appears to bind to IL-6R on a site remote from the IL-6 binding domain. Concordantly, HSJ633 selectively inhibits STAT3 phosphorylation while preserving the activation of cytoprotective AKT, p38, and ERK 1/2. In vivo, in a murine model of LPS-induced PTB, HSJ633 reduces inflammation in gestational and fetal tissues, preserves the integrity of fetal organs, and improves the survival of neonatal progeny when administered before and after the induction of labor by an inflammatory stimulus. Relevantly, the pharmacological inhibition of STAT3 in mice is sufficient to prevent PTB. Findings reveal first-in-class efficacy of a small peptide inhibitor of IL-6R, namely HSJ633, in impeding the inflammatory cascade associated with PTB and mitigating adverse neonatal outcomes.

Upfront treatment for patients with advanced high-grade serous ovarian cancer (HGSOC) includes a multi-hour cytoreductive surgery. Although the procedure is necessary for maximal tumor cytoreduction, understanding of the biology of systemic and intratumoral responses induced by surgical cytoreduction is limited. Through analysis of matched tumor and normal tissues and peripheral blood collected at multiple time points during cytoreductive surgery in patients with HGSOC, we demonstrate that surgery leads to rapid induction of systemic inflammatory response and activation of inflammatory signaling in the tumor and normal tissue, with interleukin-6 emerging as a dominant inflammatory pathway. A parallel study in a syngeneic murine HGSOC model recapitulated these findings and demonstrated accelerated tumor growth in response to surgery. This study highlights the previously unappreciated impact of specimen collection timing on the tumor signaling networks and provides insights into stress pathways activated by surgery, generating rationale for perioperative therapeutic interventions to reduce protumorigenic effects.

Pancreatic adenocarcinoma (PDAC) is a highly aggressive neoplasm characterized by limited therapeutic options, particularly in the realm of immunotherapy. This study aims to improve prognosis prediction to guide therapeutic decision-making, and to identify novel targets for immunotherapy of PDAC. We conducted Cox and LASSO regression analyses to develop immune-related gene signature and corresponding nomogram, and the robustness of these signatures was demonstrated using multiple approaches. Additionally, CIBERSORT, ESTIMATE, and xCell algorithms were utilized to assess immune cell infiltration, with experimental validation performed though qPCR. An immune-related gene signature consisting of 18 genes, and the prognostic nomogram was established with superior performance compared to the conventional staging system. Key parameters incorporated into the nomogram included the gene signature, tumor stage, and postoperative treatment. Patients identified as high-risk exhibited an anti-inflammatory tumor microenvironment, characterized by an increase in M2-like tumor-associated macrophages and heightened tumor purity. Notably, the expression of interleukin 6 receptor (IL6R) in PDAC was predominantly derived from macrophages and was significantly associated with patient survival outcomes. Furthermore, attenuated IL-6/IL-6R signaling was found to promote M2-like macrophage differentiation. This study successfully established an immune-related gene signature and a robust nomogram for predicting clinical outcomes in patients with PDAC. Furthermore, we identified IL6R as a promising target for future immunotherapeutic strategies.

Background/Objectives: Charcot first described ALS in 1869, but the specific mechanisms that mediate the disease pathology are still not clear. Intense research efforts have provided insight into unique neuroanatomical regions, specific neuronal populations and genetic associations for ALS and other neurodegenerative diseases; however, the experimental results also suggest a convergence of these events to common toxic pathways. We propose that common toxic pathways can be therapeutically targeted, and this intervention will be effective in slowing progression and improving patient quality of life. Here, we focus on understanding the role of IL6 trans-signaling in ALS disease processes. Methods: We leveraged unique mouse models of IL6 trans-signaling that we developed that recapitulate the production of active sIL6R in a genotypic and quantitative fashion observed in humans. Given that the SOD1 transgenic mouse is one of the most highly studied and characterized models of ALS, we bred SOD1G93A mice with IL6R trans-signaling mice to determine how enhanced trans-signaling influenced symptom onset and pathological processes, including neuromuscular junction (NMJ) denervation, glial activation and motoneuron (MN) survival. Results: The results indicate that in animals with enhanced trans-signaling, symptom onset and pathological processes were accelerated, suggesting a role in disease modification. Administration of an IL6R functional blocking antibody failed to alter accelerated symptom onset and disease progression. Conclusions: Future work to investigate the site-specific influence of enhanced IL6 trans-signaling and the tissue-specific bioavailability of potential therapeutics will be necessary to identify targets for precise therapeutic interventions that may limit disease progression in the 60% of ALS patients who inherit the common Il6R Asp358Ala variant.

Accumulating evidence suggests that cancer-associated fibroblast (CAF) macroautophagy/autophagy is crucial in tumor development and may be a therapeutic target for pancreatic ductal adenocarcinoma (PDAC). However, the role of CAF autophagy during immune surveillance and cancer immunotherapy is unclear. The present study revealed that the inhibition of CAF autophagy suppresses in vivo tumor development in immune-deficient xenografts. This deletion compromises anti-tumor immunity and anti-tumor efficacy both in vitro and in vivo by upregulating CD274/PDL1 levels in an immune-competent mouse model. A block in CAF autophagy reduced the production of IL6 (interleukin 6), disrupting high desmoplastic TME and decreasing USP14 expression at the transcription level in pancreatic cancer cells. We further identify USP14 as the post-translational factor responsible for downregulating CD274 expression by removing K63 linked-ubiquitination at the K280 residue. Finally, chloroquine diphosphate-loaded mesenchymal stem cell (MSC)-liposomes, by accurately targeting CAFs, inhibited CAF autophagy, improving the efficacy of immunochemotherapy to combat pancreatic cancer.Abbreviation: AIR: adaptive immune resistance; ATRA: all-trans-retinoicacid; CAF: cancer-associated fibroblast; CD274/PDL1: CD274 molecule; CM: conditioned medium; CQ: chloroquine diphosphate; CyTOF: Mass cytometry; FGF2/bFGF: fibroblast growth factor 2; ICB: immune checkpoint blockade; IF: immunofluorescence; IHC: immunohistochemistry; IP: immunoprecipitation; MS: mass spectrometer; MSC: mesenchymal stem cell; PDAC: pancreatic ductal adenocarcinoma; TEM: transmission electron microscopy; TILs: tumor infiltrating lymphocytes; TME: tumor microenvironment; USP14: ubiquitin specific peptidase 14.

The coronavirus disease 2019 (COVID-19) spread rapidly and claimed millions of lives worldwide. Acute respiratory distress syndrome (ARDS) is the major cause of COVID-19-associated deaths. Due to the limitations of current drugs, developing effective therapeutic options that can be used rapidly and safely in clinics for treating severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infections is necessary. This study aims to investigate the effects of two food-extracted immunomodulatory agents, ajoene-enriched garlic extract (AGE) and cruciferous vegetables-extracted sulforaphane (SFN), on anti-inflammatory and immune responses in a SARS-CoV-2 acute lung injury mouse model.

Mutations of the CBP/p300 histone acetyltransferase (HAT) domain can be linked to leukemic transformation in humans, suggestive of a checkpoint of leukocyte compartment sizes. Here, we examined the impact of reversible inhibition of this domain by the small-molecule A485. We found that A485 triggered acute and transient mobilization of leukocytes from the bone marrow into the blood. Leukocyte mobilization by A485 was equally potent as, but mechanistically distinct from, granulocyte colony-stimulating factor (G-CSF), which allowed for additive neutrophil mobilization when both compounds were combined. These effects were maintained in models of leukopenia and conferred augmented host defenses. Mechanistically, activation of the hypothalamus-pituitary-adrenal gland (HPA) axis by A485 relayed shifts in leukocyte distribution through corticotropin-releasing hormone receptor 1 (CRHR1) and adrenocorticotropic hormone (ACTH), but independently of glucocorticoids. Our findings identify a strategy for rapid expansion of the blood leukocyte compartment via a neuroendocrine loop, with implications for the treatment of human pathologies.

Preterm birth preceded by spontaneous preterm labour often occurs in the clinical setting of sterile intra-amniotic inflammation (SIAI), a condition that currently lacks treatment.

Muscle-residing regulatory T cells (Tregs) control local tissue integrity and function. However, the molecular interface connecting Treg-based regulation with muscle function and regeneration remains largely unexplored. Here, we show that exercise fosters a stable induction of highly functional muscle-residing Tregs with increased expression of amphiregulin (Areg), EGFR, and ST2. Mechanistically, we find that mice lacking IL6Rα on T cells (TKO) harbor significant reductions in muscle Treg functionality and satellite and fibro-adipogenic progenitor cells, which are required for muscle regeneration. Using exercise and sarcopenia models, IL6Rα TKO mice demonstrate deficits in Tregs, their functional maturation, and a more pronounced decline in muscle mass. Muscle injury models indicate that IL6Rα TKO mice have significant disabilities in muscle regeneration. Treg gain of function restores impaired muscle repair in IL6Rα TKO mice. Of note, pharmacological IL6R blockade in WT mice phenocopies deficits in muscle function identified in IL6Rα TKO mice, thereby highlighting the clinical implications of the findings.

Monocytes are heterogeneous innate effector leukocytes generated in the bone marrow and released into circulation in a CCR2-dependent manner. During infection or inflammation, myelopoiesis is modulated to rapidly meet the demand for more effector cells. Danger signals from peripheral tissues can influence this process. Herein we demonstrate that repetitive TLR7 stimulation via the epithelial barriers drove a potent emergency bone marrow monocyte response in mice. This process was unique to TLR7 activation and occurred independently of the canonical CCR2 and CX3CR1 axes or prototypical cytokines. The monocytes egressing the bone marrow had an immature Ly6C-high profile and differentiated into vascular Ly6C-low monocytes and tissue macrophages in multiple organs. They displayed a blunted cytokine response to further TLR7 stimulation and reduced lung viral load after RSV and influenza virus infection. These data provide insights into the emergency myelopoiesis likely to occur in response to the encounter of single-stranded RNA viruses at barrier sites.

Clonal hematopoiesis (CH) increases the risk of atherosclerotic cardiovascular disease possibly due to increased plaque inflammation. Human studies suggest that limitation of interleukin-6 (IL-6) signaling could be beneficial in people with large CH clones, particularly in TET2 CH. Here we show that IL-6 receptor antibody treatment reverses the atherosclerosis promoted by Tet2 CH, with reduction of monocytosis, lesional macrophage burden and macrophage colony-stimulating factor 1 receptor (CSF1R) expression. IL-6 induces expression of Csf1r in Tet2-deficient macrophages through enhanced STAT3 binding to its promoter. In mouse and human Tet2-deficient macrophages, IL-6 increases CSF1R expression and enhances macrophage survival. Treatment with the CSF1R inhibitor PLX3397 reversed accelerated atherosclerosis in Tet2 CH mice. Our study demonstrates the causality of IL-6 signaling in Tet2 CH accelerated atherosclerosis, identifies IL-6-induced CSF1R expression as a critical mechanism and supports blockade of IL-6 signaling as a potential therapy for CH-driven cardiovascular disease.

Newborns are unable to reach the adult-level humoral immune response partly due to the potent immunoregulatory role of IL-10. Increased IL-10 production by neonatal B cells has been attributed to the larger population of IL-10-producting CD43+ B-1 cells in neonates. Here, we show that neonatal mouse CD43- non-B-1 cells also produce substantial amounts of IL-10 following B cell antigen receptor (BCR) activation. In neonatal mouse CD43- non-B-1 cells, BCR engagement activated STAT5 under the control of phosphorylated forms of signaling molecules Syk, Btk, PKC, FAK, and Rac1. Neonatal STAT5 activation led to IL-6 production, which in turn was responsible for IL-10 production in an autocrine/paracrine fashion through the activation of STAT3. In addition to the increased IL-6 production in response to BCR stimulation, elevated expression of IL-6Rα expression in neonatal B cells rendered them highly susceptible to IL-6-mediated STAT3 phosphorylation and IL-10 production. Finally, IL-10 secreted from neonatal mouse CD43- non-B-1 cells was sufficient to inhibit TNF-α secretion by macrophages. Our results unveil a distinct mechanism of IL-6-dependent IL-10 production in BCR-stimulated neonatal CD19+CD43- B cells.

The association between cancer and autoimmune disease is unexplained, exemplified by T cell large granular lymphocytic leukemia (T-LGL) where gain-of-function (GOF) somatic STAT3 mutations correlate with co-existing autoimmunity. To investigate whether these mutations are the cause or consequence of CD8+ T cell clonal expansions and autoimmunity, we analyzed patients and mice with germline STAT3 GOF mutations. STAT3 GOF mutations drove the accumulation of effector CD8+ T cell clones highly expressing NKG2D, the receptor for stress-induced MHC-class-I-related molecules. This subset also expressed genes for granzymes, perforin, interferon-γ, and Ccl5/Rantes and required NKG2D and the IL-15/IL-2 receptor IL2RB for maximal accumulation. Leukocyte-restricted STAT3 GOF was sufficient and CD8+ T cells were essential for lethal pathology in mice. These results demonstrate that STAT3 GOF mutations cause effector CD8+ T cell oligoclonal accumulation and that these rogue cells contribute to autoimmune pathology, supporting the hypothesis that somatic mutations in leukemia/lymphoma driver genes contribute to autoimmune disease.

Interleukin-6 (IL-6), a pleiotropic cytokine with both degenerative and regenerative properties, is necessary and sufficient to provoke perioperative neurocognitive disorders after aseptic trauma in mice. IL-6 initiates its actions after binding to either membrane-bound IL-6 receptor α (mIL-6Rα) through classical signalling, or soluble IL-6 receptor (IL-6R) through trans-signalling; both signalling pathways require the transducer gp130. We investigated the site and type of IL-6 signalling that pertains in a tibial fracture aseptic trauma model of perioperative neurocognitive disorder.

Malignant brain tumours are the cause of a disproportionate level of morbidity and mortality among cancer patients, an unfortunate statistic that has remained constant for decades. Despite considerable advances in the molecular characterization of these tumours, targeting the cancer cells has yet to produce significant advances in treatment. An alternative strategy is to target cells in the glioblastoma microenvironment, such as tumour-associated astrocytes. Astrocytes control multiple processes in health and disease, ranging from maintaining the brain's metabolic homeostasis, to modulating neuroinflammation. However, their role in glioblastoma pathogenicity is not well understood. Here we report that depletion of reactive astrocytes regresses glioblastoma and prolongs mouse survival. Analysis of the tumour-associated astrocyte translatome revealed astrocytes initiate transcriptional programmes that shape the immune and metabolic compartments in the glioma microenvironment. Specifically, their expression of CCL2 and CSF1 governs the recruitment of tumour-associated macrophages and promotes a pro-tumourigenic macrophage phenotype. Concomitantly, we demonstrate that astrocyte-derived cholesterol is key to glioma cell survival, and that targeting astrocytic cholesterol efflux, via ABCA1, halts tumour progression. In summary, astrocytes control glioblastoma pathogenicity by reprogramming the immunological properties of the tumour microenvironment and supporting the non-oncogenic metabolic dependency of glioblastoma on cholesterol. These findings suggest that targeting astrocyte immunometabolic signalling may be useful in treating this uniformly lethal brain tumour.