IL-1 Blockade Research Articles

Immunogenicity and efficacy of CNA25 as a potential whole-cell vaccine against systemic candidiasis

Disseminated fungal infections account for ~1.5 million deaths per year worldwide, and mortality may increase further due to a rise in the number of immunocompromised individuals and drug-resistance fungal species. Since an approved antifungal vaccine is yet to be available, this study explored the immunogenicity and vaccine efficacy of a DNA polymerase mutant strain of Candida albicans. CNA25 is a pol32ΔΔ strain that exhibits growth defects and does not cause systemic candidiasis in mice. Immunized mice with live CNA25 were fully protected against C. albicans and C. parapsilosis but partially against C. tropicalis and C. glabrata infections. CNA25 induced steady expression of TLR2 and Dectin-1 receptors leading to a faster recognition and clearance by the immune system associated with the activation of protective immune responses mostly mediated by neutrophils, macrophages, NK cells, B cells, and CD4+ and CD8+ T cells. Molecular blockade of Dectin-1, IL-17, IFNγ, and TNFα abolished resistance to reinfection. Altogether, this study suggested that CNA25 collectively activates innate, adaptive, and trained immunity to be a promising live whole-cell vaccine against systemic candidiasis.

Interleukin 17 as a central component of the pathogenesis of pain associated with immunoinflammatory process: A new “target” of pharmacotherapy

Modern pathogenetic therapy of inflammatory rheumatic diseases (IRD) is aimed not only at reducing disease activity (although achieving remission and low disease activity remains the main goal of treatment), but also at eliminating as quickly and completely as possible the main symptoms that cause a decrease in the quality of life of patients. Particular importance is attached to effective control of chronic pain – the main and most distressing manifestation of IRD. To solve this problem, the pathogenesis of chronic pain in IRD continues to be actively studied, aimed at finding new ”targets” of pharmacotherapy. Thus, the role of central sensitization (CS) and comorbid fibromyalgia in the formation of clinical manifestations of IRD is now clearly proven. Signs of CS, depending on the instrument of its detection, are determined in 20–40% of patients with rheumatoid arthritis (RA), psoriatic arthritis (PsA) and axial spondyloarthritis (AxSpA).Interleukin (IL) 17 plays a fundamental role in the development of chronic pain in IIRD. This cytokine takes a leading position in the development of the ”cytokine cascade”, inducing the synthesis of various cytokines and chemokines, as well as chemotaxis and activation of neutrophils and T cells. Induction of synthesis of inflammatory mediators (including prostaglandin E2) determines the role of IL-17 in activation of nociceptors and their sensitization. IL-17 also takes an active part in neuroimmune interactions by activating glia cells and affecting receptors present on the membrane of neurons of the posterior horns of the spinal cord. This defines the role of IL-17 as one of the inductors of CS development. Pharmacologic blockade of IL-17 is a known pathway to suppress the activity of IIRPs such as PsA and AxSpA. However, this mechanism also allows for significant effects on chronic pain. In particular, the IL-17 inhibitor ixekizumab has shown high analgesic potential in a series of studies in PsA and AxSpA (SPIRIT-P1 and SPIRIT-P2, COAST V and COAST W). It is important to note that this drug demonstrated a very rapid analgesic effect: pain intensity was significantly reduced already 7 days after the first injection. These data suggest a specific effect of ixekizumab on the nociceptive system, independent of the anti-inflammatory effect. This fact allows us to consider ixekizumab as a drug of choice for the treatment of patients with PsA and AxSpA who experience severe pain and have signs of CS and fibromyalgia.

Interleukin 33 supports squamous cell carcinoma growth via a dual effect on tumour proliferation, migration and invasion, and T cell activation

Interleukin (IL)-33 is an important cytokine in the tumour microenvironment; it is known to promote the growth and metastasis of solid cancers, such as gastric, colorectal, ovarian and breast cancer. Our group demonstrated that the IL-33/ST2 pathway enhances the development of squamous cell carcinoma (SCC). Conversely, other researchers have reported that IL-33 inhibits tumour progression. In addition, the crosstalk between IL-33, cancer cells and immune cells in SCC remains unknown. The aim of this study was to investigate the effect of IL-33 on the biology of head and neck SCC lines and to evaluate the impact of IL-33 neutralisation on the T cell response in a preclinical model of SCC. First, we identified epithelial and peritumoural cells as a major local source of IL-33 in human SCC samples. Next, in vitro experiments demonstrated that the addition of IL-33 significantly increased the proliferative index, motility and invasiveness of SCC-25 cells, and downregulated MYC gene expression in SCC cell lines. Finally, IL-33 blockade significantly delayed SCC growth and led to a marked decrease in the severity of skin lesions. Importantly, anti-IL-33 monoclonal antibody therapy increase the percentage of CD4+IFNγ+ T cells and decreased CD4+ and CD8+ T cells secreting IL-4 in tumour-draining lymph nodes. Together, these data suggest that the IL-33/ST2 pathway may be involved in the crosstalk between the tumour and immune cells by modulating the phenotype of head and neck SCC and T cell activity. IL-33 neutralisation may offer a novel therapeutic strategy for SCC.

OA36 Systemic juvenile idiopathic arthritis: the Great Ormond Street Hospital experience (2005-2021)

Abstract Background/Aims sJIA is a complex, systemic-inflammatory disorder. The proposed biphasic model of disease suggests that unchecked early auto-inflammation driven by increased innate immune activation, including dysregulation of IL-1 and IL-6, may lead to a state of chronic T-lymphocyte activation. This leads to an unfavourable balance of T-effector and regulatory cells that can drive chronic arthritis over time. It is therefore unsurprising that there is growing evidence to support instigation of IL-1 or IL-6 blockade as first-line treatment in sJIA. Early cytokine antagonist therapy could abrogate development of a population of these arthritis-causing T-cells to avert chronic systemic inflammation and arthritis. Aims: 1) describe clinical presentations, therapeutic interventions, complications, and remission rates at different time-points over disease course in sJIA; 2) identify potential therapeutic signals in patients with sJIA who received biologic treatment early in disease course compared to those who did not. Methods Retrospective review of all patients with sJIA at GOSH (02/10/05-21/10/21, inclusive). Data collected: patient demographics; clinical and laboratory data; treatment history; adverse events. Time-points for data collection: at diagnosis; 3-months; 1-year; last follow-up. Descriptive statistics reported as median and range or IQR for continuous variables, and as absolute frequencies and percentages for categorical variables. Comparisons of quantitative variables were made by Mann-Whitney U-test. Categorical data were compared using Fisher’s exact test (p-values<0.05 were considered significant). Results A total of 76 children (female n = 40, 53%) were diagnosed with sJIA; median age was 4.5 years (range 0.6-14.1). A biologic disease modifying anti-rheumatic drug (bDMARD) alone was commenced as first-line treatment in 28% (n = 21/76) of the cohort; however, at last review, 84% (n = 64/76) had received treatment with a bDMARD. Clinically inactive disease (CID) was achieved by 88% (n = 67/76) of the cohort at last review; however, only 32% (24/76) achieved treatment-free CID. At 1-year follow-up, CID was achieved in a significantly greater proportion of children who received treatment with a bDMARD within 3-months of diagnosis compared to those who did not (90% versus 53%, p = 0.002). Conclusion We propose a UK treatment-algorithm for children diagnosed with sJIA (see figure). We recommend reappraisal of the current NHS England commissioning policy. Disclosure C. Foley: None. D. McKenna: None. K. Gallagher: None. K. McLellan: None. H. Alkhdher: None. S. Lacassagne: None. E. Moraitis: None. C. Papadopoulou: None. C. Pilkington: None. M. Al Obaidi: None. D. Eleftheriou: None. P. Brogan: None.

Lkb1 orchestrates γδ T-cell metabolic and functional fitness to control IL-17-mediated autoimmune hepatitis.

γδ T cells play a crucial role in immune surveillance and serve as a bridge between innate and adaptive immunity. However, the metabolic requirements and regulation of γδ T-cell development and function remain poorly understood. In this study, we investigated the role of liver kinase B1 (Lkb1), a serine/threonine kinase that links cellular metabolism with cell growth and proliferation, in γδ T-cell biology. Our findings demonstrate that Lkb1 is not only involved in regulating γδ T lineage commitment but also plays a critical role in γδ T-cell effector function. Specifically, T-cell-specific deletion of Lkb1 resulted in impaired thymocyte development and distinct alterations in γδ T-cell subsets in both the thymus and peripheral lymphoid tissues. Notably, loss of Lkb1 inhibited the commitment of Vγ1 and Vγ4 γδ T cells, promoted the maturation of IL-17-producing Vγ6 γδ T cells, and led to the occurrence of fatal autoimmune hepatitis (AIH). Notably, clearance of γδ T cells or blockade of IL-17 significantly attenuated AIH. Mechanistically, Lkb1 deficiency disrupted metabolic homeostasis and AMPK activity, accompanied by increased mTORC1 activation, thereby causing overactivation of γδ T cells and enhanced apoptosis. Interestingly, activation of AMPK or suppression of mTORC1 signaling effectively inhibited IL-17 levels and attenuated AIH in Lkb1-deficient mice. Our findings highlight the pivotal role of Lkb1 in maintaining the homeostasis of γδ T cells and preventing IL-17-mediated autoimmune diseases, providing new insights into the metabolic programs governing the subset determination and functional differentiation of thymic γδ T cells.

Outcome of Tocilizumab Treatment in Febrile Neutropenic Children with Severe Sepsis/Septic Shock in a Single-Center Retrospective Case Series.

To assess the efficacy of an IL-6 blockade with tocilizumab on treatment outcome of severe sepsis/septic shock in children with febrile neutropenia. We performed a retrospective study of febrile neutropenic patients younger than 18 years old who developed severe sepsis/septic shock at a single medical center between November 2022 and October 2023. Seven patients with febrile neutropenia complicated with severe sepsis/septic shock were identified. Four of seven patients received tocilizumab in addition to standard of care. The median IL-6 level before administration of tocilizumab was 14,147 pg/mL (range: 672-30,509 pg/mL). All four patients successfully recovered from severe sepsis/septic shock. Three of seven patients received standard of care without tocilizumab. IL-6 levels were checked intwo2 patients, with a median of 1514.5 (range: 838-2191). Only one of three (33%) patients without tocilizumab therapy made a full recovery from severe sepsis/septic shock. The mortality rate was higher in patients without tocilizumab therapy compared to patients with tocilizumab therapy (67% vs. 0%). Administration of tocilizumab reduced mortality of severe sepsis/septic shock in children with febrile neutropenia. However, it warrants confirmation with a larger number of patients and a longer follow-up.

Giant Cell Arteritis.

Giant cell arteritis (GCA) is the most common primary vasculitis and is associated with potential bilateral blindness. Neither clinical nor laboratory evidence is simple and unequivocal for this disease, which usually requires rapid and reliable diagnosis and therapy. The ophthalmologist should consider GCA with the following ocular symptoms: visual loss or visual field defects, transient visual disturbances (amaurosis fugax), diplopia, eye pain, or new onset head or jaw claudication. An immediate ophthalmological examination with slit lamp, ophthalmoscopy, and visual field, as well as color duplex ultrasound of the temporal artery should be performed. If there is sufficient clinical suspicion of GCA, corticosteroid therapy should be initiated immediately, with prompt referral to a rheumatologist/internist and, if necessary, temporal artery biopsy should be arranged. Numerous developments in modern imaging with colour duplex ultrasonography, MRI, and PET-CT have the potential to compete with the classical, well-established biopsy of a temporal artery. Early determination of ESR and CRP may support RZA diagnosis. Therapeutically, steroid-sparing immunosuppression with IL-6 blockade or methotrexate can be considered. These developments have led to a revision of both the classification criteria and the diagnostic and therapeutic recommendations of the American College of Rheumatologists and the European League against Rheumatism, which are summarised here for ophthalmology.

Anti-inflammatory effects and related mechanisms in vitro and in vivo of Hedychium coccineum rhizome essential oil

Ethnopharmacological relevanceHedychium coccineum rhizome is an anti-inflammatory ethnomedicine used to remedy inflammation-related swelling and bronchial asthma. Aim of the studyThe study aimed to analyze the phytochemical constituents of H. coccineum rhizome essential oil (EO) and evaluate its in vitro and in vivo anti-inflammatory effects and underlying mechanisms. Materials and methodsPhytochemical constituents of H. coccineum rhizome EO were analyzed using GC-FID/MS. In RAW264.7 macrophages induced by LPS, blockade of PGE2, NO, IL-1β, IL-6, and TNF-α secretion by H. coccineum rhizome EO was measured, and then Western blot, qRT-PCR, and immunofluorescent staining were used to evaluate its underlying mechanisms. Moreover, we used the xylene-induced ear edema model for testing anti-inflammatory potential in vivo and examined auricular swelling as well as tissue and serum contents of IL-1β, IL-6, and TNF-α. ResultsEO's main components were E-nerolidol (40.5%), borneol acetate (24.8%), spathulenol (4.5%), linalool (3.8%), elemol (3.5%), and borneol (3.4%). In RAW264.7 cells stimulated by LPS, EO downregulated the expression of pro-inflammatory enzyme (iNOS and COX-2) genes and proteins, thereby suppressing pro-inflammatory mediators (NO and PGE2) secretion. Simultaneously, it reduced TNF-α, IL-1β, and IL-6 release by downregulating their mRNA expression. Besides, H. coccineum EO attenuated LPS-stimulated activation of NF-κB (by reducing IκBα phosphorylation and degradation to inhibit NF-κB nuclear translocation) and MAPK (by downregulating JNK, p38, and ERK phosphorylation). In xylene-induced mouse ear edema, EO relieved auricular swelling and lowered serum and tissue levels of TNF-α, IL-1β, and IL-6. ConclusionsH. coccineum EO had powerful in vivo and in vitro anti-inflammatory effects by inhibiting MAPK and NF-κB activation. Hence, H. coccineum EO should have great potential for application in the pharmaceutical field as a novel anti-inflammatory agent.

Abstract 4048: Comparative effects of SOS1 inhibitor with IL-6 blockade and their combination on Kras mutant lung cancer

Abstract Despite recent advances in treatment, lung adenocarcinoma (LUAD), particularly harboring Kras mutations, is still the leading cause of cancer mortality. We and others have previously shown that Kras mutant LUAD development and progression is associated with intrinsic inflammatory characteristics driven by epithelial NF-κB activation, increased levels of IL-6 (which is transcriptionally regulated by NF-κB), and activation of the IL-6-responsive transcription factor STAT3. We further demonstrated that IL-6 blockade suppresses lung tumorigenesis by reducing tumor-associated immunosuppressive myeloid populations and induction of a T cell-mediated anti-tumor response. The recent discovery of a novel SOS1-targeting compound (BI-3406) that selectively inhibits the KRAS-SOS1 interaction and leads to anti-proliferative activity has opened up the possibility to more efficiently inhibit Kras mutant lung cancer. Accordingly, in this study we used a mouse model of KRAS mutant LUAD (CC-LR) to compare the therapeutic effects of BI-3406, IL-6 blockade, and MEK inhibitor (trametinib). We also tested whether inhibiting IL-6 driven pro-tumor immune response would provide a synergistic/additive response and increase the tumor inhibitory effect of either BI-3406 or Trametinib. Eight cohorts of 14-week-old female mice were treated with either anti-IL-6 antibody, trametinib, or BI-3406, or their combinations for 4 weeks, and their tumor burden was compared at the end of study. We observed a significant reduction in tumor multiplicity and tumor area in response to all monotherapies, with the highest effect in the trametinib treated cohort. Neither combination therapy led to decreased tumor burden over either of the monotherapy regimens in our model. However, we observed an increase in T helper 1 and CD8 T expressing IFNγ cell populations in the cohort treated with Trametinib and anti-IL-6 Ab combination. In addition, using qPCR analysis of RNA extracted from the whole lung we found a higher expression of IFNγ along with a reduced expression of IL-10 in the cohort treated with BI-3406 and anti-IL-6 Ab combination. This suggests that these combinations may have the potential to improve the anti-tumor immune responses. Hence, we conclude that further adjustment of the treatment regimen (doses, interval, duration) may allow to obtain improved anti-tumor efficacy with these combinations. Overall, this study provides a new research perspective for the improvement of currently available treatment modalities for patients with Kras mutant lung cancer that could be further explored preclinically. Citation Format: Katherine E. Larsen, Maria J. Arredondo Sancristobal, Melody Zarghooni, Avantika Krishna, Maria T. Grimaldo, Nastaran Karimi, Arnav Gaitonde, Michael J. Clowers, Seyed Javad Moghaddam. Comparative effects of SOS1 inhibitor with IL-6 blockade and their combination on Kras mutant lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4048.

Abstract 627: IL-6 signaling via JAK/STAT axis influences PIM1 expression in renal cell carcinoma

Abstract Renal cell carcinoma (RCC) is a top ten malignancy in the U.S. Overexpression of the proviral integration site for moloney murine leukemia virus 1 (PIM1) kinase is associated with poor clinical outcomes in RCC patients. PIM1 is a constitutively active serine/threonine kinase promoting cell proliferation, apoptosis resistance, invasion, and migration. The mechanisms underlying PIM1 expression and its function in RCC are not fully delineated. IL-6 is a pleiotropic cytokine that activates the JAK/STAT signaling cascade. High serum IL-6 levels are associated with the poor prognosis of RCC patients and may contribute to RCC invasion and metastasis. STAT3/5 binds directly to the PIM1 promoter inducing PIM1 expression. An IL-6/STAT3/PIM1 axis exists in pancreatic and breast cancer. We previously reported that PIM1 is overexpressed in a panel of human RCC cell lines relative to normal and immortalized renal proximal tubule epithelial cells. We also identified that RCC cells secrete IL-6. Our prior studies suggest that differential expression of PIM1 may be linked to autocrine IL-6 signaling. We thus hypothesize that an IL-6/JAK/STAT pathway regulates the expression of PIM1 in RCC. To understand how IL-6 signaling through the JAK/STAT pathway may regulate PIM1 expression in RCC cells, we examined whether IL-6 blockade using anti-IL-6 antibody or tocilizumab, would modulate PIM1 expression. Similarly, we assessed whether ruxolitinib, and LLL12, a STAT3 inhibitor could regulate PIM1 expression. We then evaluated the effect of ruxolitinib treatment on cell viability. In RCC cell lines, IL-6 blockade through either anti-IL-6 antibody or tocilizumab was sufficient to decrease PIM1 protein levels. Treatment with ruxolitinib leads to a dose and time-dependent decrease in PIM1 levels. Incubation with a STAT3 inhibitor also resulted in decreased PIM1 levels in RCC cells. Treatment with ruxolitinib also appears to decrease cell viability in a dose-dependent manner. These results suggest that differential expression of PIM1 in RCC may be linked to autocrine IL-6 signaling via a JAK/STAT/PIM1 axis. Multiple FDA-approved agents are available that target this pathway. Further investigation is required to determine the efficacy of these agents in pre-clinical models and even clinical trials. Citation Format: Kimberly S. Meza, Kimberly Seymour, Sheldon L. Holder. IL-6 signaling via JAK/STAT axis influences PIM1 expression in renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 627.

Mice, myeloid cells, and dengue: a new model for unraveling vascular leakage mysteries.

Severe dengue is thought to be caused by an excessive host immune response. To study the pathogenesis of severe dengue, we developed a novel model using LysM Cre+Ifnarflox/flox mice carrying depleted Ifnar expression only in subsets of murine myeloid cells. Although dengue virus (DENV) clinical isolates were not virulent in LysM Cre+Ifnarflox/flox mice, mouse-adapted DV1-5P7Sp and DV3P12/08P4Bm, which were obtained by passaging the spleen or bone marrow of mice, demonstrated 100% lethality with severe vascular leakage in the liver and small intestine. DV1-5P7Sp and DV3P12/08P4Bm harbored five and seven amino acid substitutions, respectively. Infection also induced neutrophil infiltration in the small intestine, and increased expression of IL-6 and MMP-8 and blockade of TNF-α signaling protected the mice, as demonstrated in a previous severe dengue mouse model using C57/BL6 mice lacking both IFN-α/β and IFN-γ receptors. Notably, the new models with DV1-5P7Sp and DV3P12/08P4Bm showed an increased proliferative capacity of the adapted viruses in the thymus and bone marrow. These observations suggest that myeloid cell infection is sufficient to trigger cytokine storm-induced vascular leakage. This model can refine the factors involved in the pathology of severe dengue leading to vascular leakage.

Combined IL6 and CCR2 blockade potentiates antitumor activity of NK cells in HPV-negative head and neck cancer

BackgroundWhile T cell-activating immunotherapies against recurrent head and neck squamous cell carcinoma (HNSCC) have shown impressive results in clinical trials, they are often ineffective in the majority of patients. NK cells are potential targets for immunotherapeutic intervention; however, the setback in monalizumab-based therapy in HNSCC highlights the need for an alternative treatment to enhance their antitumor activity.MethodsSingle-cell RNA sequencing (scRNA-seq) and TCGA HNSCC datasets were used to identify key molecular alterations in NK cells. Representative HPV-positive ( +) and HPV-negative ( −) HNSCC cell lines and orthotopic mouse models were used to validate the bioinformatic findings. Changes in immune cells were examined by flow cytometry and immunofluorescence.ResultsThrough integration of scRNA-seq data with TCGA data, we found that the impact of IL6/IL6R and CCL2/CCR2 signaling pathways on evasion of immune attack by NK cells is more pronounced in the HPV − HNSCC cohort compared to the HPV + HNSCC cohort. In orthotopic mouse models, blocking IL6 with a neutralizing antibody suppressed HPV − but not HPV + tumors, which was accompanied by increased tumor infiltration and proliferation of CD161+ NK cells. Notably, combining the CCR2 chemokine receptor antagonist RS504393 with IL6 blockade resulted in a more pronounced antitumor effect that was associated with more activated intratumoral NK cells in HPV − HNSCC compared to either agent alone.ConclusionsThese findings demonstrate that dual blockade of IL6 and CCR2 pathways effectively enhances the antitumor activity of NK cells in HPV-negative HNSCC, providing a novel strategy for treating this type of cancer.

Comparative efficacy and therapeutic positioning of biologics in hidradenitis suppurativa: A systematic review with network meta-analysis of randomised trials.

Background Hidradenitis suppurativa (HS) is a challenging inflammatory skin condition. Recently, many different biologics have been tested for HS, but the paucity of head-to-head comparative trials makes it difficult to determine the real value of each biological intervention. We aimed to determine the relative efficacy among biologics in treating moderate-to-severe HS throughout a network meta-analysis (NMA) and, to identify which pathogenetic pathways may be the most appropriate to target. Methods We comprehensively identified studies in 3 databases and clinicaltrials.gov. The eligibility criteria included randomised controlled trials (RCTs) reporting data on the efficacy of moderate-to-severe HS. Results The NMA comprised 13 studies comprising 14 interventions on 2,748 participants in the network. The NMA showed the odds of achieving the clinical response were significantly superior with adalimumab (RR: 0.37, 95% CI = 0.06-0.63), adalimumab QW (RR: 0.63, 95% CI = 0.43-0.87), MAB1p (RR: 1.33, 95% CI = 0.03-3.12), secukinumab (RR: 0.25, 95% CI = 0.11-0.47) and secukinumabQ2W (RR: 0.24, 95% CI = 0.1-0.46) compared to placebo. Conclusion Based on the NMA, inhibiting tumour necrosis factor (TNF)-a with adalimumab appears to be the best strategy, followed by the blockade of IL--17 with secukinumab. Data for bimekizumab and CJM112 are promising. Infliximab has inconsistent clinical response, and more data are necessary to confirm this molecule as a potential third-line therapy in HS. The blockade of IL-23 and CD5a pathways is not relevant, or at least the current evidence is insufficient to recommend further investigation of guselkumab, risankizumab, and vilobelimab in phase III trials.

Single-cell analysis of psoriasis resolution demonstrates an inflammatory fibroblast state targeted by IL-23 blockade

Biologic therapies targeting the IL-23/IL-17 axis have transformed the treatment of psoriasis. However, the early mechanisms of action of these drugs remain poorly understood. Here, we perform longitudinal single-cell RNA-sequencing in affected individuals receiving IL-23 inhibitor therapy. By profiling skin at baseline, day 3 and day 14 of treatment, we demonstrate that IL-23 blockade causes marked gene expression shifts, with fibroblast and myeloid populations displaying the most extensive changes at day 3. We also identify a transient WNT5A+/IL24+ fibroblast state, which is only detectable in lesional skin. In-silico and in-vitro studies indicate that signals stemming from these WNT5A+/IL24+ fibroblasts upregulate multiple inflammatory genes in keratinocytes. Importantly, the abundance of WNT5A+/IL24+ fibroblasts is significantly reduced after treatment. This observation is validated in-silico, by deconvolution of multiple transcriptomic datasets, and experimentally, by RNA in-situ hybridization. These findings demonstrate that the evolution of inflammatory fibroblast states is a key feature of resolving psoriasis skin.

BHLHE40 drives protective polyfunctional CD4 T cell differentiation in the female reproductive tract against Chlamydia.

The protein basic helix-loop-helix family member e40 (BHLHE40) is a transcription factor recently emerged as a key regulator of host immunity to infections, autoimmune diseases and cancer. In this study, we investigated the role of Bhlhe40 in protective T cell responses to the intracellular bacterium Chlamydia in the female reproductive tract (FRT). Mice deficient in Bhlhe40 exhibited severe defects in their ability to control Chlamydia muridarum shedding from the FRT. The heightened bacterial burdens in Bhlhe40-/- mice correlated with a marked increase in IL-10-producing T regulatory type 1 (Tr1) cells and decreased polyfunctional CD4 T cells co-producing IFN-γ, IL-17A and GM-CSF. Genetic ablation of IL-10 or functional blockade of IL-10R increased CD4 T cell polyfunctionality and partially rescued the defects in bacterial control in Bhlhe40-/- mice. Using single-cell RNA sequencing coupled with TCR profiling, we detected a significant enrichment of stem-like T cell signatures in Bhlhe40-deficient CD4 T cells, whereas WT CD4 T cells were further down on the differentiation trajectory with distinct effector functions beyond IFN-γ production by Th1 cells. Altogether, we identified Bhlhe40 as a key molecular driver of CD4 T cell differentiation and polyfunctional responses in the FRT against Chlamydia.

Abstract B025: IL-1 blockade prevents cardiac toxicity and improves immunotherapy efficacy in mouse models of pancreatic cancer

Abstract Background: Immune checkpoint blockers (ICBs) have revolutionized cancer treatment, but they are often associated with severe immune related adverse events (irAEs). These severe irAEs are more often seen in patients with obesity or concomitantly treated with cytotoxic therapies. Methods: We aimed to understand the mechanisms of ICB-induced irAEs, in the context of obesity and ICB/chemotherapy combinations. We developed a mouse model of cardiac irAEs, which is the most fatal type of irAE in ICB-treated cancer patients, with clinically relevant features: (i) an ICB-resistant cancer (pancreatic ductal adenocarcinoma or PDAC), (ii) obesity induced with high-fat diets, and (iii) a combination treatment of ICB (α-PD1 + α-CTLA4) and chemotherapy (FOLFIRINOX). Results: Our FDA and single institution retrospective analyses indicate that patients treated with ICB had greater relative risk of developing myocarditis as compared to other anti-cancer therapies. Mice with orthotopic PDAC and obesity developed irAEs after treatment with ICB and chemotherapy as compared to chow diet. These irAEs recapitulated those observed in patients with cancer and obesity, including cardiac dysfunction consistent with myocarditis, cardiac fibrosis, and increased circulating levels of interleukin-1 beta (IL-1b). IL-1β blockade prevented myocarditis and reduced cardiac fibrosis after immunotherapy. Importantly, IL-1β blockade also enhanced the anti-tumor effects of ICB + FOLFIRINOX combination therapy, and increased mouse survival. Conclusions: We developed a translationally relevant 'triple hit' mouse model and discovered that IL-1β mediates ICB-induced cardiotoxicity. In addition, we found that IL-1β blockers, which are already used in the clinic for cardiology indications, both reduce adverse events and simultaneously enhance the antitumor effects triggered by immunotherapy. Citation Format: Nilesh P. Talele, Heena Kumra, Igor L. Gomes-Santos, Sylvie Roberge, William W. Ho, Patrick Andersson, Sampurna Chatterjee, Marie Siwicki, Dan G. Duda, Mikael J. Pittet, Dai Fukumura, Rakesh K. Jain. IL-1 blockade prevents cardiac toxicity and improves immunotherapy efficacy in mouse models of pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B025.

Assessment of IL-9/IL-9R Expression and Therapeutic Effect of IL-9 Blockade in a Mouse Breast Cancer Model

Background and Hypothesis:IL-9 is a cytokine produced by TH9, mast cells, and innate lymphoid cells. IL-9 acts on the IL-9 receptor (IL-9R) and is primarily involved in parasitic immunity and allergic inflammation. IL-9 has anti-tumor effects in solid tumors such as melanoma. IL-9 also has pro-tumor effects in various cancer types, including lung cancer. Previous studies have shown that increased serum IL-9 in breast cancer patients correlates with tumor metastasis. Therefore, we hypothesize that IL9 signaling contributes to breast tumor metastasis and blocking IL-9 may have a therapeutic benefit in reducing metastasis.
 Methods:Mice were orthotopically implanted with 4T1 breast tumor cells. Organs were harvested after 28 days. Total RNA was extracted and expression of IL-9/IL-9R and leukocyte markers were assessed. To test therapeutic effect of IL-9 blockade, tumor-bearing mice were treated with anti-IL-9 monoclonal antibody (αIL-9). Tumor sizes were monitored every 4 days. On day 28, tumor tissue was harvested and weighed. Lung was also harvested and stained with H&E for metastasis analysis.
 Results:Primary tumor growth was not altered by αIL-9. The effect of αIL-9 treatment on lung metastasis is pending pathological analysis. We further examined IL-9R to define how tumor burden alters expression of IL-9 and IL-9R across tissues. Lymphoid organs, such as the thymus, spleen, and inguinal lymph node (inguinal LN), exhibit high levels of IL-9R. Additionally, the small intestine is notably enriched with IL-9R. The thymus exhibited the highest IL-9 expression.
 Conclusion and Future Directions:Although blockade of IL-9 did not impact tumor growth in this model, this will be examined in other models to confirm the findings. Further research is needed to investigate the potential therapeutic benefits of αIL-9 with other established therapies. We observed that there is altered IL-9 and IL-9R expression in tumor bearing mice and the physiological significance of that finding remains to be determined.

IL-1 Family Blockade in Cytokine Storm Syndromes.

Interleukin-1 is a prototypic proinflammatory cytokine that is elevated in cytokine storm syndromes (CSSs), such as secondary hemophagocytic lymphohistiocytosis (sHLH) and macrophage activation syndrome (MAS). IL-1 has many pleotropic and redundant roles in both innate and adaptive immune responses. Blockade of IL-1 with recombinant human interleukin-1 receptor antagonist has shown efficacy in treating CSS. Recently, an IL-1 family member, IL-18, has been demonstrated to be contributory to CSS in autoinflammatory conditions, such as in inflammasomopathies (e.g., NLRC4 mutations). Anecdotally, recombinant IL-18 binding protein can be of benefit in treating IL-18-driven CSS. Lastly, another IL-1 family member, IL-33, has been postulated to contribute to CSS in an animal model of disease. Targeting of IL-1 and related cytokines holds promise in treating a variety of CSS.

TLR9-independent CD8+ T cell responses in hepatic AAV gene transfer through IL-1R1-MyD88 signaling

Upon viral infection of the liver, CD8+ Tcell responses may be triggered despite the immune suppressive properties that manifest in this organ. We sought to identify pathways that activate responses to a neoantigen expressed in hepatocytes, using adeno-associated viral (AAV) gene transfer. It was previously established that cooperation between plasmacytoid dendritic cells (pDCs), which sense AAV genomes by Toll-like receptor 9 (TLR9), and conventional DCs promotes cross-priming of capsid-specific CD8+ Tcells. Surprisingly, we find local initiation of a CD8+ Tcell response against antigen expressed in ∼20% of murine hepatocytes, independent of TLR9 or type I interferons and instead relying on IL-1 receptor 1-MyD88 signaling. Both IL-1α and IL-1β contribute to this response, which can be blunted by IL-1 blockade. Upon AAV administration, IL-1-producing pDCs infiltrate the liver and co-cluster with XCR1+ DCs, CD8+ Tcells, and Kupffer cells. Analogous events were observed following coagulation factor VIII gene transfer in hemophilia A mice. Therefore, pDCs have alternative means of promoting anti-viral Tcell responses and participate in intrahepatic immune cell networks similar to those that form in lymphoid organs. Combined TLR9 and IL-1 blockade may broadly prevent CD8+ T responses against AAV capsid and transgene product.

Proinflammatory cytokines driving cardiotoxicity in COVID-19.

Cardiac involvement is common in patients hospitalized with COVID-19 and correlates with an adverse disease trajectory. While cardiac injury has been attributed to direct viral cytotoxicity, serum-induced cardiotoxicity secondary to serological hyperinflammation constitutes a potentially amenable mechanism that remains largely unexplored. To investigate serological drivers of cardiotoxicity in COVID-19 we have established a robust bioassay that assessed the effects of serum from COVID-19 confirmed patients on human embryonic stem cell (hESC)-derived cardiomyocytes. We demonstrate that serum from COVID-19 positive patients significantly reduced cardiomyocyte viability independent of viral transduction, an effect that was also seen in non-COVID-19 acute respiratory distress syndrome (ARDS). Serum from patients with greater disease severity led to worse cardiomyocyte viability and this significantly correlated with levels of key inflammatory cytokines, including IL-6, TNF-α, IL1-β, IL-10, CRP, and neutrophil to lymphocyte ratio with a specific reduction of CD4+ and CD8+ cells. Combinatorial blockade of IL-6 and TNF-α partly rescued the phenotype and preserved cardiomyocyte viability and function. Bulk RNA sequencing of serum-treated cardiomyocytes elucidated specific pathways involved in the COVID-19 response impacting cardiomyocyte viability, structure, and function. The observed effects of serum-induced cytotoxicity were cell-type selective as serum exposure did not adversely affect microvascular endothelial cell viability but resulted in endothelial activation and a procoagulant state. These results provide direct evidence that inflammatory cytokines are at least in part responsible for the cardiovascular damage seen in COVID-19 and characterise the downstream activated pathways in human cardiomyocytes. The serum signature of patients with severe disease indicates possible targets for therapeutic intervention.