MAPK p38/Ulk1 pathway inhibits autophagy and induces IL-1β expression in hepatic stellate cells

Background: In the past, hepatic stellate cells (HSCs) were considered to be noninflammatory cells and contribute to liver fibrosis by producing extracellular matrix. Recently, it was found that HSCs can also secrete cytokines and chemokines and therefore participate in hepatic inflammation. Autophagy participates in many immune response processes in immune cells. It is unclear whether autophagy is involved in inflammatory cytokine induction in HSCs. Methods: MAPK p38, Ulk1 phosphorylation and the Ulk1-Atg13 complex were analyzed in HSC-T6 cells after LPS treatment. The relationship between autophagy inhibition and inflammation was investigated in primary rat HSCs. Results: We discovered that LPS inhibited autophagy through MAPK p38. The activation of MAPK p38 induced Ulk1 phosphorylation, which disrupted the Ulk1-Atg13 complex and therefore inhibited autophagy. Furthermore, in primary rat HSCs, we demonstrated that autophagy inhibition regulated IL-1β induction, which depended on the MAPK p38/Ulk1 pathway. Conclusions: Our results reveal a continuous signaling pathway, MAPK p38-Ulk1 phosphorylation-Ulk1/Atg13 disruption, which inhibits autophagy and induces IL-1β expression in HSCs.

Differential recognition and cytokine induction by the peptidorhamnomannan from Sporothrix brasiliensis and S. schenckii

Sporotrichosis is a deep mycosis caused by dimorphic species of the genus Sporothrix, with differences in pathogenicity between S. schenckii and S. brasiliensis species. Recently, it was discovered that the cell wall peptidorhamnomannan (PRM) of Sporothrix spp . is a pathogen associated molecular pattern (PAMP). Interestingly, S. brasiliensis PRM has additional unknown rhamnose residues. We hypothesize that the structural differences of Sporothrix spp PRMs impact the host's immune response and may explain the severity of sporotrichosis caused by S. brasiliensis. Here we demonstrate that S. brasiliensis yeasts and its PRM (S.b PRM) induced a strong inflammatory response in human PBMCs, with high production of TNF-α, IL-6 and IL-1β and induction of T-helper cytokines IFN-γ, IL-17 and IL-22. In contrast, S. schenckii yeasts and its PRM induced higher concentrations of interleukin-1 receptor antagonist (IL-1Ra), which resulted in low production of T-helper cytokines such as IL-17 and IL-22. CR3 and dectin-1 were required for cytokine induction by both PRMs, while TLR2 and TLR4 were required for the response of S.s PRM and S.b PRM, respectively. IL-1β and IL-1α production induced by S. brasiliensis yeasts and S.b PRM were dependent on inflammasome and caspase-1 activation. S. schenckii and S.s PRM were able to induce IL-1β independent of ROS. In conclusion, these findings improve our understanding of the pathogenesis of Sporothrix spp. by reporting differences of immunological responses induced by S. schenckii and S. brasiliensis. The study also opens the gateway for novel treatment strategies targeting local inflammation and tissue destruction induced by S. brasiliensis infection through IL-1 inhibition.

SARS-CoV-2 Nsp5 Activates NF-κB Pathway by Upregulating SUMOylation of MAVS

The COVID-19 is an infectious disease caused by SARS-CoV-2 infection. A large number of clinical studies found high-level expression of pro-inflammatory cytokines in patients infected with SARS-CoV-2, which fuels the rapid development of the disease. However, the specific molecular mechanism is still unclear. In this study, we found that SARS-CoV-2 Nsp5 can induce the expression of cytokines IL-1β, IL-6, TNF-α, and IL-2 in Calu-3 and THP1 cells. Further research found that Nsp5 enhances cytokine expression through activating the NF-κB signaling pathway. Subsequently, we investigated the upstream effectors of the NF-κB signal pathway on Nsp5 overexpression and discovered that Nsp5 increases the protein level of MAVS. Moreover, Nsp5 can promote the SUMOylation of MAVS to increase its stability and lead to increasing levels of MAVS protein, finally triggering activation of NF-κB signaling. The knockdown of MAVS and the inhibitor of SUMOylation treatment can attenuate Nsp5-mediated NF-κB activation and cytokine induction. We identified a novel role of SARS-CoV-2 Nsp5 to enhance cytokine production by activating the NF-κB signaling pathway.

Development of a Predictive Model for Cytokine Release Syndrome to Inform Risk Stratification and CRS Management Following Immunotherapy

Background: Cytokine release syndrome (CRS) is a potentially life-threatening toxicity caused by immune activation. CRS can be triggered non-specifically by T-cell engaging therapies. Risk factors for CRS are expected to be disease-specific and prediction of an individual patient's CRS risk is not currently possible. Glofitamab is a T-cell engaging bispecific antibody targeting CD20 and CD3 with a novel 2:1 format that has shown promising efficacy with a manageable safety profile in NP30179 (NCT03075696), an ongoing Phase I/II dose finding study evaluating glofitamab in patients with relapsed or refractory non-Hodgkin lymphoma (NHL). While CRS is observed with glofitamab, cases typically occur with grade 1/2 severity (per ASTCT, Lee et al 2019) with most occurrences confined to the first cycle of therapy. Data from NP30179 were used to develop a model to predict the occurrence of Grade ≥ 2 CRS after the first glofitamab dose to enable stratification of patients according to risk of CRS with possible future implications for intensity of monitoring for those at low risk. Methods: Glofitamab was administered intravenously over 4 to 8 hours as a fixed or step-up dosing regimen, as previously described (Hutchings, et al JCO 2021). Non-overlapping training and validation data sets were defined; the training data set included patients with aggressive (n=165) or indolent NHL (n=31) who received a first dose of 0.6-25 mg, and the model validation data set (n=51; 35 aggressive NHL) included patients who received a 2.5mg first dose. The primary outcome was defined as Grade ≥2 CRS in the week after the first glofitamab dose, and included 65 events (n=58 training, n=7 validation). In the training data set we evaluated the association between the dose, putative risk factors (including demographics, medical history, disease characteristic variables, baseline laboratory values) and the occurrence of CRS. Univariate and multivariate models were applied in a stratified cross-validation setting to assess the most predictive and stable combination of risk factors. Baseline and on-treatment cytokine levels were analyzed via ELISA in a subset of patients (n=89). Results: The temporal pattern of CRS occurrences revealed that the vast majority of first CRS events occur after the first dose of glofitamab. To predict the occurrence of Grade ≥ 2 CRS after the first glofitamab dose, a multivariate model was developed to include glofitamab first dose and a combined risk score, termed the "CRS risk score" (CRSRS), which is the weighted sum of binarized risk factor values at baseline (Figure). The predictive ability was tested in the validation data set in which the incidence of Grade ≥ 2 CRS was 14% (7/51). A low risk group (CRSRS <5.0) was identified to be 60% of the test cohort with patients within this group having only 5% chance (Negative Predictive Value=0.95, SE=0.03) of experiencing a Grade ≥2 CRS. Induction of cytokines, including IL-6 and TNFα, was observed upon treatment with glofitamab and peak magnitude of cytokine induction was associated with CRS incidence and severity. Cytokine induction was evident by end of glofitamab infusion and peak levels of TNFα were observed by mid-infusion. CRSRS and TNFα induction were evaluated together for the subset of patients with cytokine data available to determine whether risk classification incorporating both metrics could refine the risk stratification of patients. Early TNFα changes were layered on top of CRSRS (at the cutoff of 5.0) such that patients with less than 1.5-fold induction were classified as low risk, and patients with more than 8-fold induction were classified as high risk. In the training data set, this decision tree approach improved the performance of prediction compared to CRSRS alone. Conclusions: A model based on 8 baseline factors allowed an accurate classification of risk for Grade ≥2 CRS upon treatment with glofitamab. Addition of TNFα induction may improve the predictive value. The predictive performance was confirmed in a separate validation data set with additional analyses in independent cohorts ongoing. The CRSRS, alone or in combination with cytokine induction, represents a tool to predict the occurrence of Grade ≥2 CRS after the first glofitamab dose to enable stratification of patients according to risk of CRS with possible future implications for the intensity of monitoring for those at low risk. K.V.K. and A.B. have contributed equally. Disclosures Belousov: F. Hoffmann-La Roche Ltd: Current Employment. Byrtek: Genentech, Inc.: Current Employment; F. Hoffmann-La Roche Ltd: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Kwan: Genentech, Inc.: Current Employment, Current equity holder in publicly-traded company. Perez-Callejo: F. Hoffmann-La Roche Ltd: Current Employment, Current equity holder in publicly-traded company. Li: Genentech, Inc.: Current Employment, Current holder of individual stocks in a privately-held company. Carlile: AstraZeneca: Current equity holder in publicly-traded company, Ended employment in the past 24 months; F. Hoffmann-La Roche Ltd: Current Employment, Current equity holder in publicly-traded company. McCall: Genentech, Inc.: Current Employment; F. Hoffmann-La Roche Ltd: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Nielsen: F. Hoffmann-La Roche Ltd: Current Employment, Current equity holder in publicly-traded company. Piccione: Genentech, Inc.: Current Employment; F. Hoffmann-La Roche Ltd: Current equity holder in publicly-traded company. OffLabel Disclosure: Glofitamab is a full-length, humanized, immunoglobulin G1 bispecific antibody with a 2:1 molecular format that facilitates bivalent binding to CD20 on B-cells, and monovalent binding to CD3 on T-cells. Glofitamab redirects T cells to engage and eliminate malignant B cells. Glofitamab is an investigational agent.

Data-driven discovery of targets for bipotent anticancer drugs identifies Estrogen Related Receptor Alpha

Drugs that kill tumors through multiple mechanisms have potential for broad clinical benefits, with a reduced propensity to resistance. We developed BipotentR, a computational approach to find cancer-cell-specific regulators that simultaneously modulate tumor immunity and another oncogenic pathway. Using tumor metabolism as proof-of-principle, BipotentR identified 38 candidate immune-metabolic regulators by combining epigenomes with bulk and single-cell tumor transcriptomes from patients. Inhibition of top candidate ESRRA (Estrogen Related Receptor Alpha) killed tumors by direct effects on energy metabolism and two immune mechanisms: (i) cytokine induction, causing proinflammatory macrophage polarization (ii) antigen-presentation stimulation, recruiting CD8+T cells into tumors. ESRRA is activated in immune-suppressive and immunotherapy-resistant tumors of many types, suggesting broad clinical relevance. We also applied BipotentR to angiogenesis and growth-suppressor pathways, demonstrating a widely applicable approach to identify drug targets that act simultaneously through multiple mechanisms. BipotentR is publicly available at http://bipotentr.dfci.harvard.edu/.

Evolutionary Profile for (Host and Viral) MLKL Indicates Its Activities as a Battlefront for Extensive Counteradaptation

Pathogen infection triggers host innate defenses which may result in the activation of regulated cell death (RCD) pathways such as apoptosis. Given a vital role in immunity, apoptotic effectors are often counteracted by pathogen-encoded antagonists. Mounting evidence indicates that programmed necrosis, which is mediated by the RIPK3/MLKL axis and termed necroptosis, evolved as a countermeasure to pathogen-mediated inhibition of apoptosis. Yet, it is unclear whether components of this emerging RCD pathway display signatures associated with pathogen conflict that are rare in combination but common to key host defense factors, namely, rapid evolution, viral homolog (virolog), and cytokine induction. We leveraged evolutionary sequence analysis that examines rates of amino acid replacement, which revealed: 1) strong and recurrent signatures of positive selection for primate and bat RIPK3 and MLKL, and 2) elevated rates of amino acid substitution on multiple RIPK3/MLKL surfaces suggestive of past antagonism with multiple, distinct pathogen-encoded inhibitors. Furthermore, our phylogenomics analysis across poxvirus genomes illuminated volatile patterns of evolution for a recently described MLKL viral homolog. Specifically, poxviral MLKLs have undergone numerous gene replacements mediated by duplication and deletion events. In addition, MLKL protein expression is stimulated by interferons in human and mouse cells. Thus, MLKL displays all three hallmarks of pivotal immune factors of which only a handful of factors like OAS1 exhibit. These data support the hypothesis that over evolutionary time MLKL functions—which may include execution of necroptosis—have served as a major determinant of infection outcomes despite gene loss in some host genomes.

Caspase-8 mutations associated with head and neck cancer differentially retain functional properties related to TRAIL-induced apoptosis and cytokine induction

The cysteine protease, caspase-8, undergoes dimerization, processing, and activation following stimulation of cells with death ligands such as TRAIL, and mediates TRAIL induction of the extrinsic apoptosis pathway. In addition, caspase-8 mediates TRAIL-induced activation of NF-κB and upregulation of immunosuppressive chemokines/cytokines, via a mechanism independent of caspase-8 catalytic activity. The gene encoding procaspase-8 is mutated in 10% of human head and neck squamous cell carcinomas (HNSCCs). Despite a paucity of experimental evidence, HNSCC-associated caspase-8 mutations are commonly assumed to be loss of function. To investigate their functional properties and phenotypic effects, 18 HNSCC-associated caspase-8 mutants were expressed in doxycycline-inducible fashion in cell line models wherein the endogenous wild-type caspase-8 was deleted. We observed that 5/8 mutants in the amino-terminal prodomain, but 0/10 mutants in the carboxyl-terminal catalytic region, retained an ability to mediate TRAIL-induced apoptosis. Caspase-8 proteins with mutations in the prodomain were defective in dimerization, whereas all ten of the catalytic region mutants efficiently dimerized, revealing an inverse relationship between dimerization and apoptosis induction for the mutant proteins. Roughly half (3/8) of the prodomain mutants and 9/10 of the catalytic region mutants retained the ability to mediate TRAIL induction of immunosuppressive CXCL1, IL-6, or IL-8. Doxycycline-induced expression of wild-type caspase-8 or a representative mutant led to an increased percentage of T and NKT cells in syngeneic HNSCC xenograft tumors. These findings demonstrate that HNSCC-associated caspase-8 mutants retain properties that may influence TRAIL-mediated apoptosis and cytokine induction, as well as the composition of the tumor microenvironment.

Monocyte-dependent co-stimulation of cytokine induction in human γδ T cells by TLR8 RNA ligands

Human Vγ9Vδ2 T cells recognize pyrophosphates produced by microbes and transformed cells and play a role in anti-infective immunity and tumor surveillance. Toll-like receptors (TLR) are pattern recognition receptors in innate immune cells which sense microbial structures including nucleic acids. Given that γδ T cells are in clinical development for application in cellular cancer immunotherapy and TLR ligands have potent adjuvant activity, we investigated the co-stimulatory role of selected TLR ligands in γδ T-cell activation. Here we have used recently described RNA ligands for TLR7 and TLR8 together with Vγ9Vδ2 T-cell specific pyrophosphate antigens to analyze the rapid cytokine induction in Vδ2 T cells as well as the accessory cell requirements. While TLR8- as well as TLR7/8-specific RNA did not induce IFN-γ in Vδ2 T cells on their own, they provided strong co-stimulation for Vδ2 T cells within peripheral blood mononuclear cells in the presence of additional T-cell receptor activation. In contrast, TLR7 ligands were ineffective. Purified γδ T cells did not directly respond to TLR8 co-stimulation but required the presence of monocytes. Further experiments revealed a critical role of IL-1β and IL-18, and to a slightly lesser extent of IL-12p70, in the co-stimulation of Vδ2 T cells by TLR8 and TLR7/8 RNA ligands. Results of intracellular cytokine expression were validated by ELISA analysis of cytokines in cell culture supernatants. The cell context-dependent adjuvant activity of TLR8 and TLR7/8 RNA ligands described here might be important for the future optimization of γδ T-cell based cancer immunotherapy.

Variable Induction of Pro-Inflammatory Cytokines by Commercial SARS CoV-2 Spike Protein Reagents: Potential Impacts of LPS on In Vitro Modeling and Pathogenic Mechanisms In Vivo

Proinflammatory cytokine production following infection with severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) is associated with poor clinical outcomes. Like SARS CoV-1, SARS CoV-2 enters host cells via its spike protein, which attaches to angiotensin-converting enzyme 2 (ACE2). As SARS CoV-1 spike protein is reported to induce cytokine production, we hypothesized that this pathway could be a shared mechanism underlying pathogenic immune responses. We herein compared the capabilities of Middle East Respiratory Syndrome (MERS), SARS CoV-1 and SARS CoV-2 spike proteins to induce cytokine expression in human peripheral blood mononuclear cells (PBMC). We observed that only specific commercial lots of SARS CoV-2 induce cytokine production. Surprisingly, recombinant SARS CoV-2 spike proteins from different vendors and batches exhibited different patterns of cytokine induction, and these activities were not inhibited by blockade of spike protein-ACE2 binding using either soluble ACE2 or neutralizing anti-S1 antibody. Moreover, commercial spike protein reagents contained varying levels of lipopolysaccharide (LPS), which correlated directly with their abilities to induce cytokine production. The LPS inhibitor, polymyxin B, blocked this cytokine induction activity. In addition, SARS CoV-2 spike protein avidly bound soluble LPS in vitro, rendering it a cytokine inducer. These results not only suggest caution in monitoring the purity of SARS CoV-2 spike protein reagents, but they indicate the possibility that interactions of SARS CoV-2 spike protein with LPS from commensal bacteria in virally infected mucosal tissues could promote pathogenic inflammatory cytokine production.

Injectable Nanoparticle-Based Hydrogels Enable the Safe and Effective Deployment of Immunostimulatory CD40 Agonist Antibodies

When properly deployed, the immune system can render deadly pathogens harmless, eradicate metastatic cancers, and provide long-lasting protection from diverse diseases. However, realizing these remarkable capabilities is inherently risky, as disruption to immune homeostasis can lead to dangerous complications and autoimmune disorders. While current research is continuously expanding the arsenal of potent immunotherapeutics, there is a technological gap when it comes to controlling when, where, and how long these drugs act on the body. Here, we explored the ability of a slow-releasing injectable hydrogel depot to reduce the problematic dose-limiting toxicities of immunostimulatory CD40 agonist antibodies (CD40a) while maintaining their potent anti-cancer efficacy. We previously described a polymer-nanoparticle (PNP) hydrogel system that is biocompatible, long lasting, and injectable, traits that we hypothesized would improve locoregional delivery of the CD40a immunotherapy. Using PET imaging, we found that hydrogels significantly improve CD40a pharmacokinetics by redistributing drug exposure to the tumor and the tumor draining lymph node (TdLN). Consistent with this altered biodistribution, hydrogel delivery significantly reduced weight loss, hepatotoxicity, and cytokine storm associated with treatment. Moreover, CD40a-loaded hydrogels were able to mediate improved local cytokine induction in the TdLN and improve treatment efficacy in both mono- and combination therapy settings in the B16F10 melanoma model. These results suggest that PNP hydrogels are a facile, drug-agnostic method to ameliorate immune-related adverse effects and explore locoregional delivery of immunostimulatory drugs.