Background: Pancreatic ductal adenocarcinoma (PDAC) is resistant to single-agent immunotherapies. To understand the mechanisms leading to the poor response to this treatment, a better understanding of the PDAC immune landscape is required. The present work aims to study the immune profile in PDAC in relationship to spatial heterogeneity of the tissue microenvironment (TME) in intact tissues. Methods: Serial section and multiplex in situ analysis were performed in 42 PDAC samples to assess gene and protein expression at single-cell resolution in the: (a) tumor center (TC), (b) invasive front (IF), (c) normal parenchyma adjacent to the tumor, and (d) tumor positive and negative draining lymph nodes (LNs). Results: We observed: (a) enrichment of T cell subpopulations with exhausted and senescent phenotype in the TC, IF and tumor positive LNs; (b) a dominant type 2 immune response in the TME, which is more pronounced in the TC; (c) an emerging role of CD47-SIRPα axis; and (d) a similar immune cell topography independently of the neoadjuvant chemotherapy. Conclusion: This study reveals the existence of dysfunctional T lymphocytes with specific spatial distribution, thus opening a new dimension both conceptually and mechanistically in tumor-stroma interaction in PDAC with potential impact on the efficacy of immune-regulatory therapeutic modalities.

In the setting of pronounced inflammation, changes in the epithelium may overlap with neoplasia, often rendering it impossible to establish a diagnosis with certainty in daily clinical practice. Here, we discuss the underlying molecular mechanisms driving tissue response during persistent inflammatory signaling along with the potential association with cancer in the gastrointestinal tract, pancreas, extrahepatic bile ducts, and liver. We highlight the histopathological challenges encountered in the diagnosis of chronic inflammation in routine practice and pinpoint tissue-based biomarkers that could complement morphology to differentiate reactive from dysplastic or cancerous lesions. We refer to the advantages and limitations of existing biomarkers employing immunohistochemistry and point to promising new markers, including the generation of novel antibodies targeting mutant proteins, miRNAs, and array assays. Advancements in experimental models, including mouse and 3D models, have improved our understanding of tissue response. The integration of digital pathology along with artificial intelligence may also complement routine visual inspections. Navigating through tissue responses in various chronic inflammatory contexts will help us develop novel and reliable biomarkers that will improve diagnostic decisions and ultimately patient treatment.

The idea that bacterial toxins are not only killers but also execute more sophisticated roles during bacteria–host interactions by acting as negotiators has been highlighted in the past decades. Depending on the toxin, its cellular target and mode of action, the final regulatory outcome can be different. In this review, we have focused on two families of bacterial toxins: genotoxins and pore-forming toxins, which have different modes of action but share the ability to modulate the host’s immune responses, independently of their capacity to directly kill immune cells. We have addressed their immuno-suppressive effects with the perspective that these may help bacteria to avoid clearance by the host’s immune response and, concomitantly, limit detrimental immunopathology. These are optimal conditions for the establishment of a persistent infection, eventually promoting asymptomatic carriers. This immunomodulatory effect can be achieved with different strategies such as suppression of pro-inflammatory cytokines, re-polarization of the immune response from a pro-inflammatory to a tolerogenic state, and bacterial fitness modulation to favour tissue colonization while preventing bacteraemia. An imbalance in each of those effects can lead to disease due to either uncontrolled bacterial proliferation/invasion, immunopathology, or both.

We recently characterized the association between DNA damage and immunoresponse in vivo in colonic mucosa of mice infected with a Salmonella Typhimurium strain expressing a genotoxin, known as typhoid toxin. In this protocol, we describe how to assess the extent and features of infiltrating macrophages by double immunofluorescence. Total macrophage population was determined using an F4/80 antibody, whereas the specific M2-like population was assessed using a CD206 antibody.

Thrombomodulin (TM) is a membrane protein with significant roles in coagulation hemostasis and immune response. Its soluble form (sTM) has recently emerged as a key biomarker for severe invasive bacterial infections, including Necrotizing Soft Tissue Infections (NSTI). While various mechanical, chemical, and enzymatic mechanisms have been linked to TM shedding, this study investigates the direct impact of bacterial stimuli on soft tissue cells as primary sources of TM release. We stimulated organotypic models, composed of fibroblast and endothelial cells, with NSTI clinical isolates and found that while Group A Streptococcus and Escherichia coli had minimal effect on TM release, Staphylococcus aureus infection triggered a significant increase of sTM levels. We further assessed whether the secreted proteins of S. aureus led to higher TM levels by increased expression, increased cell toxicity or direct cleavage of TM from the endothelial cell membrane. To investigate these mechanisms, we performed in vitro stimulations of endothelial monolayers with secreted proteins of two S. aureus isolates differing in their agr-system functionality. Our results indicate that S. aureus' agr-regulated proteins induce TM shedding by direct cleavage from the cell membrane, an effect that was inhibited by metalloproteinase inhibitors. Stimulation with the pore-forming protein α-toxin showed similar results, suggesting a potential involvement of ADAM10 in TM cleavage. Additionally, we observed that other agr-regulated proteins can cleave TM directly. Altogether, this study reveals a pathogen-specific mechanism for TM release during S. aureus invasive infection, contributing to its elevated plasma levels and providing deeper insights into the pathophysiology of NSTI.