Transition to Practice Among Diverse Early Professional Athletic Trainers.

CONTEXT: Early professional (EP) Athletic Trainers (ATs) may encounter adjustments and develop individual identities to master a new role, which can be difficult while transitioning from student to autonomous professional. Previous literature lacks content about the transition to practice of credentialed ATs who identify as Black, Indigenous, or People of Color (BIPOC). OBJECTIVE: Identify challenges during professional education and transition to practice of EP ATs identifying as BIPOC. DESIGN: Qualitative study. SETTING: Virtual interview. PATIENTS OR OTHER PARTICIPANTS: 15 recently credentialed athletic trainers (13 female, 2 male, age=26.73±1.41 years, experience=13.80±4.03 months) who self-identified as BIPOC. DATA COLLECTION AND ANALYSIS: We used an interview guide, validated by peer (n=1) and expert (n=3) review, to structure data collection sessions. Data saturation guided recruitment and we obtained theoretical redundancy after the final interview. We analyzed data with a phenomenological approach and used multianalyst triangulation (n=2) and peer review (n=2) as credibility strategies. RESULTS: Every participant mentioned they were victims of microaggressions during either their professional preparation and/or work environment because of their race. Those who chose to report the incidents felt a lack of support from work supervisors, faculty, peers, and preceptors. Participants noted a preference for racially concordant mentoring to facilitate talking to someone who could better understand BIPOC EPs' experiences. Participants also relied on outside support from friends and family as coping mechanisms. Finally, participants experienced perceived incompetency from supervisors due to their limited work experience as early professionals and felt their professional preparation was stunted by the COVID-19 pandemic. CONCLUSIONS: Participants perceived that their transition to practice was stressful due to microaggressions encountered, a lack of support, and a lack of racially concordant mentoring. Diversity, equity, and inclusion training should be included in athletic training education and workspaces to assist in providing more welcoming environments for BIPOC EP ATs and students.

Asah2 Represses the p53-Hmox1 Axis to Protect Myeloid-Derived Suppressor Cells from Ferroptosis.

Myeloid-derived suppressor cells (MDSCs) are immune suppressive cells that massively accumulate under pathological conditions to suppress T cell immune response. Dysregulated cell death contributes to MDSC accumulation, but the molecular mechanism underlying this cell death dysregulation is not fully understood. In this study, we report that neutral ceramidase (N-acylsphingosine amidohydrolase [ASAH2]) is highly expressed in tumor-infiltrating MDSCs in colon carcinoma and acts as an MDSC survival factor. To target ASAH2, we performed molecular docking based on human ASAH2 protein structure. Enzymatic inhibition analysis of identified hits determined NC06 as an ASAH2 inhibitor. Chemical and nuclear magnetic resonance analysis determined NC06 as 7-chloro-2-(3-chloroanilino)pyrano[3,4-e][1,3]oxazine-4,5-dione. NC06 inhibits ceramidase activity with an IC50 of 10.16-25.91 µM for human ASAH2 and 18.6-30.2 µM for mouse Asah2 proteins. NC06 induces MDSC death in a dose-dependent manner, and inhibition of ferroptosis decreased NC06-induced MDSC death. NC06 increases glutathione synthesis and decreases lipid reactive oxygen species to suppress ferroptosis in MDSCs. Gene expression profiling identified the p53 pathway as the Asah2 target in MDSCs. Inhibition of Asah2 increased p53 protein stability to upregulate Hmox1 expression to suppress lipid reactive oxygen species production to suppress ferroptosis in MDSCs. NC06 therapy increases MDSC death and reduces MDSC accumulation in tumor-bearing mice, resulting in increased activation of tumor-infiltrating CTLs and suppression of tumor growth in vivo. Our data indicate that ASAH2 protects MDSCs from ferroptosis through destabilizing p53 protein to suppress the p53 pathway in MDSCs in the tumor microenvironment. Targeting ASAH2 with NC06 to induce MDSC ferroptosis is potentially an effective therapy to suppress MDSC accumulation in cancer immunotherapy.

p50 suppresses cytotoxic T lymphocyte effector function to regulate tumor immune escape and response to immunotherapy.

BACKGROUND: NF-κB is a key link between inflammation and cancer. Previous studies of NF-κB have largely focused on tumor cells, and the intrinsic function of NF-κB in T cells in tumor development and response to immunotherapy is largely unknown. We aimed at testing the hypothesis that NF-κB1 (p50) activation in T cells underlies human colon cancer immune escape and human cancer non-response to anti-PD-1 immunotherapy. METHODS: We screened NF-κB activation in human colon carcinoma and used mouse models to determine p50 function in tumor cells and immune cells. RNA-Seq was used to identify p50 target genes. p50 binding to target gene promoters were determined by electrophoresis mobility shift assay and chromatin immunoprecipitation. A p50 activation score was generated from gene expression profiling and used to link p50 activation to T-cell activation and function pre-nivolumab and post-nivolumab immunotherapy in human patients with cancer. RESULTS: p50 is the dominant form of NF-κB that is highly activated in immune cells in the human colorectal carcinoma microenvironment and neighboring non-neoplastic colon epithelial cells. Tumor cell intrinsic p50 signaling and T-cell intrinsic p50 signaling exert opposing functions in tumor growth control in vivo. Deleting Nfkb1 in tumor cells increased whereas in T cells decreased tumor growth in preclinical mouse models. Gene expression profiling identified Gzmb as a p50 target in T cells. p50 binds directly to a previously uncharacterized κB sequence at the Gzmb promoter in T cells, resulting in repression of Gzmb expression in tumor-infiltrating cytotoxic T lymphocytes (CTLs) to induce a dysfunctional CTL phenotype to promote tumor immune escape. p50 activation is inversely correlated with both GZMB expression and T-cell tumor infiltration in human colorectal carcinoma. Furthermore, nivolumab immunotherapy decreased p50 activation and increased GZMB expression in human patients with melanoma. CONCLUSIONS: Inflammation activates p50 that binds to the Gzmb promoter to repress granzyme B expression in T cells, resulting in CTL dysfunction to confer tumor immune escape and decreased response to anti-PD-1 immunotherapy.

Expression profiles and function of IL6 in polymorphonuclear myeloid-derived suppressor cells.

IL6 is an inflammatory cytokine with pleiotropic functions in both immune and nonimmune cells, and its expression level is inversely correlated with disease prognosis in patients with cancer. However, blocking IL6 alone has only yielded minimal efficacy in human cancer patients. We aimed at defining IL6 expression profiles under inflammatory conditions and cancer, and elucidating the mechanism underlying IL6 intrinsic signaling in colon carcinoma. We report here that colonic inflammation induces IL6 expression primarily in the CD11b+Ly6G+Ly6Clo polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) in colon. Although both tumor cells, T cells and myeloid cells all express IL6, PMN-MDSCs are the primary cell type that express IL6 in colon carcinoma, suggesting that IL6 up-regulation is a response to inflammation in colon epithelium and tumor microenvironment. Furthermore, we determined that IL6 activates STAT3 to up-regulate DNMT1 and DNMT3b expression in colon tumor cells, thereby revealing an epigenetic mechanism that mediates the IL6-STAT3 signaling pathway in colon carcinoma. Surprisingly, knocking out IL6 in colon tumor cells did not significantly alter tumor growth in WT mice. Conversely, IL6-sufficient colon and pancreatic tumor grow at similar rate in WT and IL6-deficient mice. However, overexpression of IL6 in colon tumor cells significantly increases tumor growth in vivo. Our findings determine that a high tumor local IL6 threshold is essential for IL6 function in colon tumor promotion and targeting the IL6-expressing PMN-MDSCs is potentially an effective approach to suppress colon tumor growth in vivo.

Autocrine IL6-Mediated Activation of the STAT3-DNMT Axis Silences the TNFα-RIP1 Necroptosis Pathway to Sustain Survival and Accumulation of Myeloid-Derived Suppressor Cells.

Although accumulation of myeloid-derived suppressor cells (MDSC) is a hallmark of cancer, the underlying mechanism of this accumulation within the tumor microenvironment remains incompletely understood. We report here that TNFα-RIP1-mediated necroptosis regulates accumulation of MDSCs. In tumor-bearing mice, pharmacologic inhibition of DNMT with the DNA methyltransferease inhibitor decitabine (DAC) decreased MDSC accumulation and increased activation of antigen-specific cytotoxic T lymphocytes. DAC-induced decreases in MDSC accumulation correlated with increased expression of the myeloid cell lineage-specific transcription factor IRF8 in MDSCs. However, DAC also suppressed MDSC-like cell accumulation in IRF8-deficient mice, indicating that DNA methylation may regulate MDSC survival through an IRF8-independent mechanism. Instead, DAC decreased MDSC accumulation by increasing cell death via disrupting DNA methylation of RIP1-dependent targets of necroptosis. Genome-wide DNA bisulfite sequencing revealed that the Tnf promoter was hypermethylated in tumor-induced MDSCs in vivo. DAC treatment dramatically increased TNFα levels in MDSC in vitro, and neutralizing TNFα significantly increased MDSC accumulation and tumor growth in tumor-bearing mice in vivo. Recombinant TNFα induced MDSC cell death in a dose- and RIP1-dependent manner. IL6 was abundantly expressed in MDSCs in tumor-bearing mice and patients with human colorectal cancer. In vitro, IL6 treatment of MDSC-like cells activated STAT3, increased expression of DNMT1 and DNMT3b, and enhanced survival. Overall, our findings reveal that MDSCs establish a STAT3-DNMT epigenetic axis, regulated by autocrine IL6, to silence TNFα expression. This results in decreased TNFα-induced and RIP1-dependent necroptosis to sustain survival and accumulation. SIGNIFICANCE: These findings demonstrate that targeting IL6 expression or function represent potentially effective approaches to suppress MDSC survival and accumulation in the tumor microenvironment.