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Understanding complex pathophysiological processes involves studying intercellular responses and phenotypes within the organ microenvironment, preserving the spatial tissue architecture. This chapter explores a practical and cost-effective method for combining techniques such as in situ immunostaining and transcriptomics analysis. These protocols are adaptable to various mRNA targets, antibodies, tissue types, and tissue fixation appealing to a wide scientific community. We demonstrate their application in studying the host response to infection with a Salmonella enterica strain producing a toxin that induces DNA breaks. Specifically, we assessed the: (i) innate immune response to DNA breaks; (ii) co-detection of Salmonella mRNA fljB with the DNA damage marker γH2AX; (iii) co-detection of mRNAs for the cell cycle arrest marker p16INK4A and the proinflammatory and anti-inflammatory cytokines, Il6 and Il10, respectively. Considering that DNA damage is one of the leading causes of oncogene- and stress-induced-senescence, these protocols can be suitable to assess the cytokine profile associated with cellular phenotype and types of DNA damage of senescent cells in situ.

In situ hybridization visualizes RNA in cells, but image analysis is complex. We present a protocol based on open-source software for automated high-content multiplex fluorescence in situ transcriptomics analysis. Steps include nuclei segmentation with a Fiji macro and quantification of up to 14 mRNA probes per image. We describe procedures for storing raw data, quality control images, and the use of a Python app to summarize all the results in one spreadsheet detailing the number of single or co-positive cells.

Introduction: Typhoid toxin-expressing Salmonella enterica causes DNA damage in the intestinal mucosa in vivo, activating the DNA damage response (DDR) in the absence of inflammation. To understand whether the tissue microenvironment constrains the infection outcome, we compared the immune response and DDR patterns in the colon and liver of mice infected with a genotoxigenic strain or its isogenic control strain.

Methods: In situ spatial transcriptomic and immunofluorescence have been used to assess DNA damage makers, activation of the DDR, innate immunity markers in a multiparametric analysis.

Result: The presence of the typhoid toxin protected from colonic bacteria-induced inflammation, despite nuclear localization of p53, enhanced co-expression of type-I interferons (IfnbI) and the inflammasome sensor Aim2, both classic features of DNA-break-induced DDR activation. These effects were not observed in the livers of either infected group. Instead, in this tissue, the inflammatory response and DDR were associated with high oxidative stress-induced DNA damage.

Conclusions: Our work highlights the relevance of the tissue microenvironment in enabling the typhoid toxin to suppress the host inflammatory response in vivo.

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. For complete details on the use and execution of this protocol, please refer to Martin et al. (2021).

Bacterial genotoxins cause DNA damage in eukaryotic cells, resulting in activation of the DNA damage response (DDR) in vitro. These toxins are produced by Gram-negative bacteria, enriched in the microbiota of inflammatory bowel disease (IBD) and colorectal cancer (CRC) patients. However, their role in infection remains poorly characterized. We address the role of typhoid toxin in modulation of the host-microbial interaction in health and disease. Infection with a genotoxigenic Salmonella protects mice from intestinal inflammation. We show that the presence of an active genotoxin promotes DNA fragmentation and senescence in vivo, which is uncoupled from an inflammatory response and unexpectedly associated with induction of an anti-inflammatory environment. The anti-inflammatory response is lost when infection occurs in mice with acute colitis. These data highlight a complex context-dependent crosstalk between bacterial-genotoxin-induced DDR and the host immune response, underlining an unexpected role for bacterial genotoxins.

Expression of typhoid toxin in Salmonella Typhimurium causes DNA damage, activating the DNA damage response (DDR), in absence of an inflammatory response in the colonic mucosa of infected mice. The anti-inflammatory effect is tissue specific and is not observed in the liver, suggesting that the local immune microenvironment modulates the DDR outcome.

To assess the role of the immune cells in the DDR outcome induced by the genotoxigenic Salmonella, we have initiated the development of an immunocompetent 3D colonic mucosal model based on a collagen matrix containing colonic fibroblasts and different subtypes of immune cells, overlayed with colonic epithelial cells.

Embedding of peripheral blood mononuclear cells in the collagen matrix did not influenced either the tissue integrity or the activation of the DDR, observed exclusively upon infection with the genotoxigenic strain. However, embedding of T cells, monocytes, or non-polarized macrophages altered the pattern of the DDR and the toxin specific effect was lost. Presence of macrophages was further associated with alteration of the epithelial layer integrity. This effect was infection-dependent, but not toxin specific.

Our data demonstrated that addition of immune cells to a 3D mucosal model altered the DDR induced by a genotoxigenic bacterium, highlighting the need to develop and optimize immunocompetent in vitro models.