BNITM researchers on the way to a skin organ model with immune cells

New insights into the interactions between skin cells, pathogens and the immune system

Mosquitoes, bugs and ticks bite humans in the skin. These vectors can infect humans with pathogens that cause diseases such as malaria, dengue fever or Chagas disease. From an immunological point of view, not much is known about the skin as an entry point for pathogens into the body. Researchers at the Bernhard Nocht Institute for Tropical Medicine (BNITM) have used a human cell culture model to study how Trypanosoma cruzi, the parasite causing Chagas disease, enters skin cells and how certain immune cells react. In the future, they hope to use a skin organ model containing immune cells for their experiments. The researchers have recently published important preliminary work for this future project in the journal PLOS Neglected Tropical Diseases.

The fluorescence image shows green skin cells, the nuclei are stained blue. A skin cell is infected with Trypanosoma cruzi, T. cruzi is stained red. The background is black.

©BNITM

Dr Thomas Jacobs, head of the Protozoa Immunology research group at the BNITM, and Dr Rosa Isela Gálvez chose to study the infection of human keratinocytes, the skin cells of the epidermis, with the parasite Trypanosoma cruzi (= T. cruzi). Triatomine bugs transmit the single-celled parasite into the skin of humans and other mammals. T. cruzi multiplies in host cells such as keratinocytes. Via the bloodstream, the parasite enters other cells and tissues, such as the muscles of the host organism. In humans, T. cruzi causes Chagas disease. Acute symptoms include local swelling at the site of skin entry. If the disease becomes chronic, organs such as the heart become enlarged and nerve cells in the gastrointestinal tract are destroyed. The disease is therefore usually fatal. Chagas disease is one of the neglected tropical diseases (NTDs) and is mainly found in Latin America.

The researchers used a novel co-culture model to study the interactions between T. cruzi-infected keratinocytes and natural killer (NK) cells. NK cells are a type of immune cell that can kill pathogen-infected cells. They chose NK cells because they are essential in the fight against T. cruzi. The scientists obtained keratinocytes from biopsies of healthy volunteers and isolated natural killer cells from blood samples taken from the same people.

Scheme of co-cultivation of keratinocytes with NK cells. 1. cultivation of keratinocytes until they reach a density suitable for the experiments. 2. infection of the keratinocytes with the unicellular parasite T. cruzi. 3. after 72 hours, the parasites have invaded the keratinocytes. 4. magnetic sorting of the different blood cells from the blood donation allows isolation of the NK cells. 5. addition of NK cells to the T. cruzi-infected keratinocytes. 6. co-cultivation of NK cells and T. cruzi-infected keratinocytes for 24 hours. 7. examination of the supernatant for soluble molecules released by the NK cells. 8. flow cytometric analysis of NK cell function, such as degranulation and activation status. 9. staining of keratinocytes and the parasite T. cruzi, followed by analysis in the Opera Phenix HCS system to determine the extent to which the NK cells have eliminated the parasites. Image created with a licensed version of Biorender. ©Kroh et al. 2024

T. cruzi infects keratinocytes in cell culture model

"As a first important step, we have shown that the T. cruzi parasite can infect keratinocytes in a cell culture model. When we move on to skin organ models, we need to make sure that the infection is successful. Otherwise, we would be wasting expensive resources," says immunologist Gálvez.

Four fluorescence images showing skin cells infected with different amounts of T. cruzi. B) Image sequence analysis of keratinocytes infected with T. cruzi Brazil. C) Bar graphs showing infection rates (left) and average number of parasites per infected keratinocyte cell (right) with different amounts of T. cruzi Brazil and T. cruzi Tulahuen parasites added. — A) Infection of keratinocytes with different amounts (MOI = multiplicity of infection; indicates the ratio of pathogen to target cells) of the parasite strain T. cruzi Brazil (TcB). T. cruzi is stained red, nuclei blue. B) Image sequence analysis of keratinocytes infected with T. cruzi Brazil. (1) image acquisition before analysis, (2) blue nuclear staining, (3) red staining of T. cruzi, (4) grey detection of nuclei, (5) cell interior of keratinocytes, (6) detection of T. cruzi (green), (7) selection of infected cells (green) and non-infected cells (red). C) Infection rates (left) and average number of parasites per infected keratinocyte cell (right) with different amounts of added parasites of the strains T. cruzi Brazil and T. cruzi Tulahuen. The infection rates of the T. cruzi Brazil strain were higher than those of the Tulahuen strain. At an MOI of 6:1, the Brazil strain infected 25 % of the keratinocytes, while the Tulahuen strain only 3%. Because of its more efficient infection and better modelling of chronic infection in humans, the researchers used the Brazil strain of T. cruzi for further experiments. ©Kroh et al. 2024

Natural killer cells recognise keratinocytes infected with T. cruzi

After successfully infecting the keratinocytes with T. cruzi, the researchers added NK cells, which were highly activated by the infected keratinocytes.

Gálvez explains: "Our studies show that keratinocytes not only serve as a reservoir for the parasite, but also play an active role in the immune response. The interaction between infected keratinocytes and NK cells leads to strong activation of the NK cells, which then release important pro-inflammatory molecules such as interferon-gamma and other cytotoxic molecules to fight the infection."

Toward an immunocompetent skin organ model

Skin organ models containing the different skin cell types already exist. However, these models still lack immune cells. The new findings of the research team led by Gálvez and Jacobs underline the importance of the skin as an immunological organ. They show how critical it is to develop a model that contains both immune cells and skin cells (i.e. an immunocompetent skin organ model). The researchers could not use the mouse model because the composition of immune cells in the skin of mice and humans is very different.

"So far, the co-culture model is the only model available for our investigations. We have now shed more light on some of the interactions between host cells, pathogens and natural killer cells. A detailed characterisation of the immune response in the skin will contribute to the development of more effective therapeutic strategies against Tryponosoma cruzi, but also against other pathogens that enter humans via the skin. For this characterisation, it is essential to establish an immunocompetent skin organ model," concludes Jacobs.