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Article
Matthias Sendler
University Medicine Greifswald
Corresponding Author
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Acute pancreatitis (AP) is an inflammatory disorder, the severe form of which is burdened with high mortality. The pathogenesis of severity-driving organ manifestations, such as respiratory and renal failure, is unknown. We used samples from 300 pancreatitis patients and an experimental model of severe acute pancreatitis (SAP) to characterize severity-dependent cytokine profiles and resident/circulating immune cell populations by flow-cytometry and real-time- fluorescence and deformability-cytometry analysis. On functional, immunolabelling and in-vivo antibody-depletion experiments we confirmed the role of inflammatory cells in pancreatitis but found neither T-cells, Ly6g+-neutrophils or granulocytic-myeloid-derived-suppressor-cells (gMDSC) but a massive mobilisation of CCR2+/CD11b+-monocytes to be responsible for lung and kidney injury during SAP. Real-time-fluorescence and deformability-cytometry analyses suggest, that the physical properties of monocytes, especially their large size, results in an obstruction of the fine capillary-systems of the lung or of the kidney glomeruli. Their selective depletion can represent a promising treatment strategy for pancreatitis as well as other inflammation-related disorders.
Keywords
Monocytes, MODS, acute pancreatitis, CCR2
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Yes there is potential Competing Interest. Oliver Otto is co-founder of Zellmechanik Dresden GmbH distributing the technology of real-time deformability cytometry.
This is a list of supplementary files associated with this preprint. Click to download.
- SupplementaryFigures.pdf
Supplement 1: Blood and serum markers of pancreatitis patients were recorded at day of hospital admission. Spearman rank order correlation tests confirmed a positive correlation for serum lactate and serum creatinine to total blood leukocyte count (a). C-reactive protein (CRP), a classical acute phase protein, also correlated with creatinine, but not with serum lactate (b). All tested chemokines (MCP-1, MIG, IP-10 and IL-8) did not correlate with the CRP level, which indicates that the leukocyte mobilisation occurs independent from the acute phase protein (c). p values are located in the upper right corner of each graph. Supplement 2: Pancreatic tissue macrophages are unaffected by a systemic monocyte depletion. Double labelling of CD68 and CCR2 indicates that only a small percentage of macrophages carried CCR2, which suggests that tissue resident CCR2- macrophages reflect the majority of cells (a). Pancreatic tissue of animals treated with anti-CCR2 antibody showed a complete loss of CCR2+ macrophages (a). Ki67 labelling, a marker of cell proliferation, showed, that macrophages start to proliferate after the induction of pancreatitis (b). Flow cytometric analysis of T-cell activation in spleen revealed a pancreatitis induced increase of CD69+, CD25+, CD25+/Foxp3+ T-helper cells and CD8+ cells in all animals, independent of a depletion of neutrophils or monocytes (c). All graphs represent 6 or more animals per group. Statistically significant differences were tested by one way ANOVA followed by Tukey’s multiple comparison test, or by Kruskal-Wallis test followed by Dunn’s multiple comparison test. A significance level of p<0.05 is marked by asterisk, rhombs indicate significant difference to the untreated control mice. Supplement 3: Serum cytokines were measured by fluorometric bead array analysis in isotype treated and CCR2-treated mice. The observed increase of pro-inflammatory cytokines IL-6 and TNFα as well as of T-cell activating cytokine IL-2 during pancreatitis was not changed in isotype or CCR2 treated mice. All graphs represent 6 or more animals each group. Supplement 4: Analysis of oxygen saturation, acid-alkaline balance and serum electrolytes in SAP mice. pO2, FO2Hb, glucose and pH were in general decreased after induction of pancreatitis whereas pCO2 and ctHb were slightly increased (a). Pancreatitis seems to disturb the acid-alkaline balance., but serum electrolytes were not affected by pancreatitis with the exception of Na+ which showed a slight increase (b). All graphs represent 6 or more animals each group. Differences were tested by one way ANOVA followed by Tukey’s multiple comparison test, or by Kruskal-Wallis test followed by Dunn’s multiple comparison test, A significance level of p<0.05 is marked by asterisk, rhombs indicate significant difference to the untreated control mice. Supplement 5: Immunofluorescent labelling of CCR2+ cells in tissue sections of lung and kidney. After induction of SAP in mice, CCR2+ macrophages (red) are located within the VE-Cadherin (white) positive areas of lung tissue. Tissue resident alveolar macrophages were labelled by CD68 (green)e and are located at the border of the alveolar space (a). In kidney sectionsCCR2+/CD68+ monocytes are detected within the glomeruli, which indicates a vascular accumulation during pancreatitis (b).
- NCOMMS2113677rs.pdf
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This is a preprint. It has not completed peer review.
Article
Matthias Sendler
University Medicine Greifswald
Corresponding Author
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 1
Posted 27 Apr, 2021
No community comments so far
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Acute pancreatitis (AP) is an inflammatory disorder, the severe form of which is burdened with high mortality. The pathogenesis of severity-driving organ manifestations, such as respiratory and renal failure, is unknown. We used samples from 300 pancreatitis patients and an experimental model of severe acute pancreatitis (SAP) to characterize severity-dependent cytokine profiles and resident/circulating immune cell populations by flow-cytometry and real-time- fluorescence and deformability-cytometry analysis. On functional, immunolabelling and in-vivo antibody-depletion experiments we confirmed the role of inflammatory cells in pancreatitis but found neither T-cells, Ly6g+-neutrophils or granulocytic-myeloid-derived-suppressor-cells (gMDSC) but a massive mobilisation of CCR2+/CD11b+-monocytes to be responsible for lung and kidney injury during SAP. Real-time-fluorescence and deformability-cytometry analyses suggest, that the physical properties of monocytes, especially their large size, results in an obstruction of the fine capillary-systems of the lung or of the kidney glomeruli. Their selective depletion can represent a promising treatment strategy for pancreatitis as well as other inflammation-related disorders.
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