Muske, Josey

ABSTRACT Cytotoxic T lymphocytes (CTLs) are known to eliminate target cells through perforin-mediated, contact-dependent killing - a process limited by the number of effector T cells despite its serial nature. CTLs likely also possess a mass killing mechanism that can eliminate target cells more efficiently. Using CAR-T cells against B7H3 present on B16 tumor targets, we demonstrate that activated T cells can also kill targets in a perforin-independent manner by releasing diffusible TNF and IFNγ capable of killing nearby targets in a paracrine fashion even if they bear no antigen. Against an unperturbed target, paracrine killing is inefficient but is enhanced by the deletion of TNFR1 signaling molecules such as TAK1, HOIP, or TBK1/IKKε. Notably, these molecules are inhibited naturally by pathogen-encoded antagonists. Expression of a microbial-encoded antagonist, such as the Yersinia -encoded YopJ that antagonizes TAK1 or Ebola-encoded VP35 that antagonizes TBK1/IKKε, alters the target cell response to these cytokines from non-lethal to lethal. We propose that target cells are inherently resistant to killing by TNF and IFNγ, but the presence of a microbial factor alters this sensitivity, providing for the selective elimination of the infected cell while minimizing harm to uninfected bystander cells. We propose the term ‘pathogen-restriction’ to describe this discriminatory mechanism. Potentially, any microbial-derived factor that crosstalks with the TNF and IFNγ signaling pathways can function as a pathogen-restriction element. GRAPHICAL ABSTRACT Short Summary T cells use TNF and IFNγ to kill target cells in a paracrine manner where target elimination is restricted by the presence of pathogen-derived factors.

Abstract Description Late-onset neonatal sepsis (LOS) is often the result of systemic bacteremia developing between 3-90 days of age, predominantly in prematurely born populations. An initial spike of IL-6 serum levels is a hallmark diagnostic tool and has been implicated as a source for the resulting cytokine storm. Yet, other than antibiotics, there is a lack of effective therapeutics targeting the overwhelming inflammatory response. We performed single-organism infections of pathogenic and commensal clinical E. coli isolates to compare the acute cytokine response in our LOS model in neonatal (postnatal day 5-7) and periweaning (postnatal day 17-20) pups. As expected, neonatal pups had increased bacterial burden and mortality during pathogenic infection compared to periweaning-age and commensal infection. An in-depth, 32-cytokine analysis of serum, liver, and spleen revealed overall elevated and tissue-specific inflammation in the neonates with a positive correlation of IL-6 serum levels to many cytokines (MCP-1, M-CSF, MIP-1β, etc.). Isolate-specific responses were assessed in an in vitro co-culture model with RAW-blue macrophages. While both isolates induced inflammatory NFκB/AP-1 activation, commensal E. coli caused elevated IL-1β while pathogenic E. coli led to significantly elevated IL-6. Both age- and pathogenic-specific inflammatory activation suggest unique signaling from the E. coli infection and the neonatal response, calling for neonatal-specific treatment options. Funding Sources Mayo Clinic Graduate School of Biomedical Sciences R01 DK134366 (Knoop) T32 DK124190-04 (Muske) Topic Categories Microbial, Parasitic, and Fungal Immunology (MPF)

Abstract Description Neonatal sepsis caused by bacteria such as Escherichia coli affects 1.3 million infants a year. The immunological responses activated in neonates during neonatal sepsis are currently thought to overwhelm the infant. Understanding where to intervene in the inflammatory response could lead to the development of therapeutics. Using Nur77-GFP reporter mice and our established animal models of neonatal sepsis, we observed γδ T cells-innate-like lymphocytes- are activated in a TCR-dependent manner in response to pathogenic bloodstream E. coli infection in neonatal mice (post-natal day 5-7). We find a subset of γδ T cells show similar levels of TCR-dependent activation of following infection with either pathogenic or commensal E. coli. In pathogenic bloodstream E. coli infection, there is a significant release of pro-inflammatory cytokines IL-17 by these CCR6+ γδ T cells in addition to IL-6 in the serum compared to serum in commensal infection. We found that in neonatal mice, CCR6+ γδ T cells have high expression of IL-6 receptor, and preliminary data suggests IL-6 blockade reduces IL-17 production. In summary, this suggests that during an E. coli systemic infection, the IL-17 producing γδ T cells are being activated by IL-6 due to higher levels of IL-6 receptor. Further work will seek to understand how γδ T cells may detrimentally contribute to neonatal sepsis outcomes. Topic Categories Microbial, Parasitic, and Fungal Immunology (MPF)

The health of the intestinal microbiota impacts tolerance at homeostasis and the strength of the inflammation response during acute bloodstream infections. A complete understanding of the feedback loop between systemic inflammation and dysregulation of the gut microbiota is necessary for inflammation management. Here we will review the many ways in which the microbiota can influence the systemic pro-inflammatory response. Short-chain fatty acids, produced through the microbial metabolism of dietary fibers, can suppress inflammation systemically; in the absence of a balanced diet or disruption of the microbiota through antibiotics, there is disrupted metabolite production, leading to systemic inflammation. Dysbiosis or inflammation in the intestines can lead to a breakdown of the sturdy intestinal–epithelial barrier. When this barrier is perturbed, immunogenic lipopolysaccharides or extracellular vesicles enter the bloodstream and induce excessive inflammation. Necessary clinical treatments, such as antifungals or antibacterials, induce microbiota dysregulation and thus increased risk of endotoxemia; though probiotics may aid in improving the microbiota health and have been shown to deflate inflammation during sepsis. Within this complicated relationship: What is in control, the dysbiotic microbiota or the systemic inflammation?

Abstract Late-onset neonatal sepsis (LOS), a leading cause of neonatal death, can result from pathogenic bacteria disseminating to the bloodstream from the intestinal microbiota. E. coli is the second most commonly isolated pathogen causing LOS. During sepsis, there are significant immune discrepancies between infants and adults, including elevated IL-6 levels in neonates, demonstrating the need for age-specific research. We have developed a murine model of LOS that uses clinical isolates of gut-resident E. coli, which results in rapid pro-inflammatory cytokine production within 6 hours and neonatal mortality within 2–3 days post-infection. Not all enteric E. coli results in inflammation and mortality, and we have classified isolates as “commensal” or “pathogenic”. TLR4−/− mice infected with pathogenic E. coli have a delayed mortality to sepsis, suggesting innate bacterial recognition is required for acute inflammation. To further determine the source of this delay, we have begun investigating signaling pathways during bacterial infection. We found the immune signaling pathway NFκB/AP1 is consistently activated in murine macrophages by commensal and pathogenic bacterial strains. However, only pathogenic bacteria induce elevated IL-6 production by murine macrophages. Interestingly, this response required live bacteria, suggesting lipopolysaccharide alone from E. coli is not sufficient to recapitulate LOS inflammation. Our future directions will explore alternative signaling pathways that are activated uniquely during pathogenic infection and induce a robust inflammatory response. Through this work, we will determine the consequence of specific pro-inflammatory cytokines in neonatal mice during pathogenic infection. Research supported by R03 DK122187.

Abstract Bloodstream infections, causing late-onset neonatal sepsis (LOS), have an incidence rate of 10% in very low birth weight (<1500 grams) infants. Despite the prevalence in neonates, the current understanding of bloodstream infections and the resulting immune response is predominately based on research in adults, which is often a complication of surgery. In contrast, sepsis in infants is often a result of the dissemination of bacteria from the gut. Additionally, observations have shown differences in the neonatal immune response, such as a decrease in gene expression associated with TLR signaling pathways, compared to adults. One question remaining in the field is the age dependent differences in pathogen detection by innate cells, including dendritic cells and macrophages. Use RAW-Blue reporter cells, and splenocytes cultured from mice at different ages, we have found disparate responses to gut-resident E. coli dependent on pathogen status of the bacteria. Specifically, we have observed differential increases in IL-6, TNFα, and IL-1β, along with NFκB activation that is dependent on live bacteria. Additionally, changes in this response were associated with age, suggesting differences in the innate immune response throughout early life. Future directions include measuring RNA expression of dendritic cells and macrophages between the age groups and exploring how the differences contribute to infection outcomes in a model of sepsis. Supported by grants from NIH (DK122187).