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1. Steamed broccoli sprouts alleviate DSS-induced inflammation and retain gut microbial biogeography in mice

   https://doi.org/10.1128/msystems.00532-23  

   Holman, Johanna M; Colucci, Louisa; Baudewyns, Dorien; Balkan, Joe; Hunt, Timothy; Hunt, Benjamin; Kinney, Marissa; Holcomb, Lola; Stratigakis, Allesandra; Chen, Grace; et al (October 2023, mSystems)

   Manichanh, Chaysavanh (Ed.)

   ABSTRACT Inflammatory bowel diseases (IBDs) are devastating conditions of the gastrointestinal tract with limited treatments, and dietary intervention may be effective and affordable for managing symptoms. Glucosinolate compounds are highly concentrated in broccoli sprouts, especially glucoraphanin (GLR), and can be metabolized by certain mammalian gut bacteria into anti-inflammatory isothiocyanates, such as sulforaphane. Gut microbiota exhibit biogeographic patterns, but it is unknown if colitis alters these or whether the location of glucoraphanin-metabolizing bacteria affects anti-inflammatory benefits. We fed specific pathogen-free C57BL/6 mice either a control diet or a 10% steamed broccoli sprout diet and gave a three-cycle regimen of 2.5% dextran sodium sulfate (DSS) in drinking water over a 34-day experiment to simulate chronic, relapsing ulcerative colitis (UC). We monitored body weight, fecal characteristics, lipocalin, serum cytokines, and bacterial communities from the luminal- and mucosal-associated populations in the jejunum, cecum, and colon. Mice fed the broccoli sprout diet with DSS treatment performed better than mice fed the control diet with DSS, and had significantly more weight gain, lower Disease Activity Index scores, lower plasma lipocalin and proinflammatory cytokines, and higher bacterial richness in all gut locations. Bacterial communities were assorted by gut location but were more homogenous across locations in the control diet + DSS mice. Importantly, our results showed that broccoli sprout feeding abrogated the effects of DSS on gut microbiota, as bacterial richness and biogeography were similar between mice receiving broccoli sprouts with and without DSS. Collectively, these results support the protective effect of steamed broccoli sprouts against dysbiosis and colitis induced by DSS. IMPORTANCEEvaluating bacterial communities across different locations in the gut provides a greater insight than fecal samples alone and provides an additional metric by which to evaluate beneficial host-microbe interactions. Here, we show that 10% steamed broccoli sprouts in the diet protects mice from the negative effects of dextran sodium sulfate-induced colitis, that colitis erases biogeographic patterns of bacterial communities in the gut, and that the cecum is not likely to be a significant contributor to colonic bacteria of interest in the DSS mouse model of ulcerative colitis. Mice fed the broccoli sprout diet during colitis performed better than mice fed the control diet while receiving DSS. The identification of accessible dietary components and concentrations that help maintain and correct the gut microbiome may provide universal and equitable approaches to IBD prevention and recovery, and broccoli sprouts represent a promising strategy. 

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2. Early life exposure to broccoli sprouts confers stronger protection against enterocolitis development in an immunological mouse model of inflammatory bowel disease

   https://doi.org/10.1128/msystems.00688-23  

   Holcomb, Lola; Holman, Johanna M; Hurd, Molly; Lavoie, Brigitte; Colucci, Louisa; Hunt, Benjamin; Hunt, Timothy; Kinney, Marissa; Pathak, Jahnavi; Mawe, Gary M; et al (December 2023, mSystems)

   Chu, Hiutung (Ed.)

   ABSTRACT Crohn’s disease (CD) is a presentation of inflammatory bowel disease (IBD) that manifests in childhood and adolescence and involves chronic and severe enterocolitis, immune and gut microbial dysregulation, and other complications. Diet and gut-microbiota-produced metabolites are sources of anti-inflammatories that could ameliorate symptoms. However, questions remain on how IBD influences biogeographic patterns of microbial location and function in the gut, how early life transitional gut communities are affected by IBD and diet interventions, and how disruption to biogeography alters disease mediation by diet components or microbial metabolites. Many studies on diet and IBD use a chemically induced ulcerative colitis model, despite the availability of an immune-modulated CD model. Interleukin-10-knockout (IL-10-KO) mice on a C57BL/6 background, beginning at age 4 or 7 weeks, were fed a control diet or one containing 10% (wt/wt) raw broccoli sprouts, which was high in the sprout-sourced anti-inflammatory sulforaphane. Diets began 7 days prior to, and for 2 weeks after inoculation withHelicobacter hepaticus,which triggers Crohn’s-like symptoms in these immune-impaired mice. The broccoli sprout diet increased sulforaphane in plasma; decreased weight stagnation, fecal blood, and diarrhea associated; and increased microbiota richness in the gut, especially in younger mice. Sprout diets resulted in some anatomically specific bacteria in younger mice and reduced the prevalence and abundance of pathobiont bacteria which trigger inflammation in the IL-10-KO mouse, for example,Escherichia coliandHelicobacter. Overall, the IL-10-KO mouse model is responsive to a raw broccoli sprout diet and represents an opportunity for more diet-host-microbiome research. IMPORTANCETo our knowledge, IL-10-KO mice have not previously been used to investigate the interactions of host, microbiota, and broccoli, broccoli sprouts, or broccoli bioactives in resolving symptoms of CD. We showed that a diet containing 10% raw broccoli sprouts increased the plasma concentration of the anti-inflammatory compound sulforaphane and protected mice to varying degrees against disease symptoms, including weight loss or stagnation, fecal blood, and diarrhea. Younger mice responded more strongly to the diet, further reducing symptoms, as well as increased gut bacterial richness, increased bacterial community similarity to each other, and more location-specific communities than older mice on the diet intervention. Crohn’s disease disrupts the lives of patients and requires people to alter dietary and lifestyle habits to manage symptoms. The current medical treatment is expensive with significant side effects, and a dietary intervention represents an affordable, accessible, and simple strategy to reduce the burden of symptoms. 

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3. Mitigating non-genetic resistance to checkpoint inhibition based on multiple states of immune exhaustion

   https://doi.org/10.1038/s41540-024-00336-6  

   Kareva, Irina; Gevertz, Jana L (December 2024, npj Systems Biology and Applications)

   Abstract Despite the revolutionary impact of immune checkpoint inhibition on cancer therapy, the lack of response in a subset of patients, as well as the emergence of resistance, remain significant challenges. Here we explore the theoretical consequences of the existence of multiple states of immune cell exhaustion on response to checkpoint inhibition therapy. In particular, we consider the emerging understanding that T cells can exist in various states: fully functioning cytotoxic cells, reversibly exhausted cells with minimal cytotoxicity, and terminally exhausted cells. We hypothesize that inflammation augmented by drug activity triggers transitions between these phenotypes, which can lead to non-genetic resistance to checkpoint inhibitors. We introduce a conceptual mathematical model, coupled with a standard 2-compartment pharmacometric (PK) model, that incorporates these mechanisms. Simulations of the model reveal that, within this framework, the emergence of resistance to checkpoint inhibitors can be mitigated through altering the dose and the frequency of administration. Our analysis also reveals that standard PK metrics do not correlate with treatment outcome. However, we do find that levels of inflammation that we assume trigger the transition from the reversibly to terminally exhausted states play a critical role in therapeutic outcome. A simulation of a population that has different values of this transition threshold reveals that while the standard high-dose, low-frequency dosing strategy can be an effective therapeutic design for some, it is likely to fail a significant fraction of the population. Conversely, a metronomic-like strategy that distributes a fixed amount of drug over many doses given close together is predicted to be effective across the entire simulated population, even at a relatively low cumulative drug dose. We also demonstrate that these predictions hold if the transitions between different states of immune cell exhaustion are triggered by prolonged antigen exposure, an alternative mechanism that has been implicated in this process. Our theoretical analyses demonstrate the potential of mitigating resistance to checkpoint inhibitors via dose modulation. 

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4. IL-12-producing cytokine factories induce precursor exhausted T cells and elimination of primary and metastatic tumors

   https://doi.org/10.1136/jitc-2024-010685  

   Nash, Amanda; DeBonis, Jonathon; Murungi, Danna; Castillo, Bertha; Kim, Boram; Hu, Fangheng; Chambers, Courtney; Nguyen, Annie; Hernandez, Andrea; Wang, Zeshi; et al (April 2025, Journal for ImmunoTherapy of Cancer)

   BackgroundCurative responses to immunotherapy require the generation of robust systemic immunity with limited toxicity. Recruitment of T cell populations such as precursor exhausted T cells (Tpex) from lymphoid tissues to tumors is a hallmark of effective treatment. However, the ability to efficiently induce this recruitment is lacking in current immunotherapy approaches. Furthermore, systemic administration of immunotherapies frequently results in dose-limiting toxicities, yielding an inadequate therapeutic window for eliciting durable responses. MethodsIn this investigation, we evaluated the safety and antitumor efficacy of locally administered interleukin 12 (IL-12) using a clinically translatable cytokine delivery platform (NCT05538624) to identify Tpex recruitment capabilities at tolerable cytokine doses. ResultsWe show IL-12 cytokine factories can effectively treat a broad spectrum of cancer types. Single-cell RNA sequencing data suggests that the antitumor efficacy seen in our studies was due to retinal pigmented epithelial cells-mIL12 treatment inducing differentiation of Tpex cells within the tumor microenvironment. When administered in combination with checkpoint therapy, IL-12 cytokine factory treatment generated systemic abscopal immunity, preventing subcutaneous tumor outgrowth in 8/9 mice with colorectal cancer and lung metastasis in mice with melanoma. Furthermore, this platform was well tolerated in a non-human primate without signs of toxicity. ConclusionsOur new immunotherapy approach provides a robust strategy for inducing Tpex recruitment and systemic immunity against a range of solid peritoneal malignancies, many incurable with current immunotherapy strategies. Notably, these features were achieved using IL-12, and by leveraging our technology, we avoided the toxicities that have prevented the translation of IL-12 to the clinic. Our findings provide a strong rationale for the clinical development of IL-12 cytokine factories. 

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5. Intracellular Salmonella delivery of an exogenous immunization antigen refocuses CD8 T cells against cancer cells, eliminates pancreatic tumors and forms antitumor immunity

   https://doi.org/10.3389/fimmu.2023.1228532  

   Raman, Vishnu; Howell, Lars M.; Bloom, Shoshana M.; Hall, Christopher L.; Wetherby, Victoria E.; Minter, Lisa M.; Kulkarni, Ashish A.; Forbes, Neil S. (October 2023, Frontiers in Immunology)

   IntroductionImmunotherapies have shown great promise, but are not effective for all tumors types and are effective in less than 3% of patients with pancreatic ductal adenocarcinomas (PDAC). To make an immune treatment that is effective for more cancer patients and those with PDAC specifically, we genetically engineered Salmonella to deliver exogenous antigens directly into the cytoplasm of tumor cells. We hypothesized that intracellular delivery of an exogenous immunization antigen would activate antigen-specific CD8 T cells and reduce tumors in immunized mice. MethodsTo test this hypothesis, we administered intracellular delivering (ID) Salmonella that deliver ovalbumin as a model antigen into tumor-bearing, ovalbumin-vaccinated mice. ID Salmonella delivers antigens by autonomously lysing in cells after the induction of cell invasion. ResultsWe showed that the delivered ovalbumin disperses throughout the cytoplasm of cells in culture and in tumors. This delivery into the cytoplasm is essential for antigen cross-presentation. We showed that co-culture of ovalbumin-recipient cancer cells with ovalbumin-specific CD8 T cells triggered a cytotoxic T cell response. After the adoptive transfer of OT-I CD8 T cells, intracellular delivery of ovalbumin reduced tumor growth and eliminated tumors. This effect was dependent on the presence of the ovalbumin-specific T cells. Following vaccination with the exogenous antigen in mice, intracellular delivery of the antigen cleared 43% of established KPC pancreatic tumors, increased survival, and prevented tumor re-implantation. DiscussionThis response in the immunosuppressive KPC model demonstrates the potential to treat tumors that do not respond to checkpoint inhibitors, and the response to re-challenge indicates that new immunity was established against intrinsic tumor antigens. In the clinic, ID Salmonella could be used to deliver a protein antigen from a childhood immunization to refocus pre-existing T cell immunity against tumors. As an off-the-shelf immunotherapy, this bacterial system has the potential to be effective in a broad range of cancer patients. 

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