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Intraperitoneal oil application causes local inflammation with depletion of resident peritoneal macrophages

Elisenda Alsina-Sanchis, Ronja Mülfarth, Iris Moll, Carolin Mogler, Juan Rodriguez-Vita, Andreas Fischer

Preprint posted on July 29, 2020 https://www.biorxiv.org/content/10.1101/2020.07.15.203885v2

Oils, used as solvents for many drugs and compounds, may cause inflammation if injected intraperitoneally.

Selected by Oindrila Bhattacharjee

Categories: immunology

Background:
Mice are treated with drugs and compounds for various purposes such as conditional alteration of a gene, depletion of cells, or recapitulating human diseases and finding their cure (Günschmann et al., 2014; Hua, Shi, Shultz, & Ren, 2018; Rosenthal & Brown, 2007). These compounds are dissolved in the respective solvents depending on their hydrophilicity and are administered via different routes such as oral gavage, intraperitoneal injection (I.P.), intravenous injection and subcutaneous injection, to maximize the effects of the treatments (Turner, Brabb, Pekow, & Vasbinder, 2011). Usually, mice treated with oil are used as controls along with the wild type mice to rule out any effects of the oil or the route of administration. This makes it essential to ensure that the mice treated with oil and the untreated wild type mice are comparable so that any significant change following treatment is attributed only to the experimental compound. It also helps in reducing the chances of side-effects, thereby improving the specificity and authenticity of the experimental system.
Alsina-Sanchis et al. study the potential of vegetal and mineral oils to induce inflammation in the peritoneum and suggest that oral gavage instead of intraperitoneal injection might be a safer way to deliver lipophilic compounds to avoid inflammation.

Key findings:
1.) Significant increase in inflammation in the omentum and the mesentery upon intraperitoneal injection of peanut and mineral oils
The authors show that the omentum of the mice treated with oil by I.P. is swollen and dark with larger blood vessels. The vessel density also increases as shown by the quantification of CD31 staining, (a marker for endothelial cells). While all the oils show inflammatory response by increased recruitment of CD11b+ cells or by the presence of foamy macrophages, the effects are most pronounced for peanut and mineral oils. Furthermore, Sirius red staining shows an increased deposition of collagen that indicates fibrosis. They observe similar changes in the mesentery too, implicating chances of peritoneal inflammation by oil injected in the peritoneum. Mice treated by oral gavage are comparable with the untreated mice with no inflammatory burden.

2.) Oils deplete tissue-resident macrophage population by causing their death
The total population of macrophages (CD45+CD11+F4/80+) does not change between untreated or oil-injected mice. But staining for Tim4, a marker for long term resident macrophages, shows a significant reduction in their number, thereby, suggesting an increased recruitment of monocyte-derived macrophages associated with depletion of the resident population.
Macrophages take up oils and perish by different mechanisms such as apoptosis, necrosis, efferocytosis and pyroptosis. The mode of death determines whether the inflammation would be resolved. The authors study the mechanisms of death in macrophages by flow cytometric analysis on the peritoneal macrophages and by staining of cell lines treated with oils. They show that taking up olive oil by macrophages leads to their death by apoptosis which aids in resolving inflammation. However, peanut oil causes pyroptosis in macrophages impeding the reduction in inflammation.

3.) Thioglycolate-induced peritonitis model and mice intraperitoneally injected with oil have comparable inflammatory burden
When bacterial peritonitis is induced in mice by treating them with thioglycolate, the authors observe that the mice receiving peanut oil by oral gavage register a 50% increase in the myeloid cell population. But when treated with peanut oil by I.P., mice do not show a significant change in the number of myeloid cells as they already exhibit a basal level of inflammation.
Taken together, the authors suggest that oral gavage is a better and safer way to administer lipophilic compounds than I.P.

What intrigued me to choose this preprint?
The authors have tried to shed light on the choice of solvents and their correct route of administration to avoid inflammation. This work may serve as a guide for those who want to treat mice for experimental purposes.

Open questions:
1.) In this work, I.P. is given for 5 consecutive days whereas oral gavage is done once. Does this treatment regime itself introduce the differential inflammation burden?
2.) Not all I.P. treatments need to be done for 5 consecutive days. I.P. can also be given once to achieve desirable effects in a context-dependent manner. In that case, how will the inflammation status be affected?
3.) I.P. results in global effects. In that case should one expect a global change in systemic inflammation?
4.) While the mechanisms underlying the death of macrophages are shown to be independent of inflammation; what properties of these oils might have led to the differential effects?

References:
Günschmann, C., Chiticariu, E., Garg, B., Hiz, M. M., Mostmans, Y., Wehner, M., & Scharfenberger, L. (2014). Transgenic mouse technology in skin biology: Inducible gene knockout in mice. Journal of Investigative Dermatology. https://doi.org/10.1038/jid.2014.213
Hua, L., Shi, J., Shultz, L. D., & Ren, G. (2018). Genetic models of macrophage depletion. In
Methods in Molecular Biology. https://doi.org/10.1007/978-1-4939-7837-3_22
Rosenthal, N., & Brown, S. (2007). The mouse ascending: Perspectives for human-disease models. Nature Cell Biology. https://doi.org/10.1038/ncb437
Turner, P. V., Brabb, T., Pekow, C., & Vasbinder, M. A. (2011). Administration of substances to laboratory animals: Routes of administration and factors to consider. Journal of the American Association for Laboratory Animal Science.

Tags: depletion, inflammation, ip, macrophage, oils, oral gavage

Posted on: 12th September 2020 , updated on: 21st September 2020

doi: https://doi.org/10.1242/prelights.24679

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  • Author's response

    Juan Rodriguez-Vita shared

    Open questions:

    1.) In this work, I.P. is given for 5 consecutive days whereas oral gavage is done once. Does this treatment regime itself introduce the differential inflammation burden?

    These are the typical application protocols for tamoxifen to induce Cre-mediated recombination. In our hands, a single oral dose is usually sufficient for proper gene recombination whereas i.p. injections have to be repeated. We do not expect that oral delivery of oil leads to any inflammation.

    2.) Not all I.P. treatments need to be done for 5 consecutive days. I.P. can also be given once to achieve desirable effects in a context-dependent manner. In that case, how will the inflammation status be affected?

    Interesting question. Indeed for some experiments a single low dose tamoxifen injection is performed (e.g. to target only a few cells for further lineage tracing). We have not performed such an experiment so we do not know whether a single oil injection leads to the same damage a five injections.

    3.) I.P. results in global effects. In that case should one expect a global change in systemic inflammation?

    We have evaluated the presence of different immune cell precursors in the blood and bone marrow but we haven’t found any effect. This would indicate that inflammation is not systemic, but localized in the peritoneum. The general state of the animals was not affected, other than showing sometimes excessive oil on the fur, the animal showed not systemic damage.

    4.) While the mechanisms underlying the death of macrophages are shown to be independent of inflammation; what properties of these oils might have led to the differential effects?

    We hypothesized that the amount of oleic acid was the critical component preventing pyroptosis and promoting apoptosis, which supports resolution of inflammation. To proof this, we have supplemented peanut oil with oleic acid and prevented peanut oil-induced pyroptosis.

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