Obstruction of Capillaries by circulating large Monocytes causes Multi Organ Dysfunction Syndrome (MODS) in Acute Pancreatitis

doi:10.21203/rs.3.rs-403798/v1

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Obstruction of Capillaries by circulating large Monocytes causes Multi Organ Dysfunction Syndrome (MODS) in Acute Pancreatitis

https://doi.org/10.21203/rs.3.rs-403798/v1

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posted 27 Apr, 2021

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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.

Endocrinology & Metabolism

Gastroenterology & Hepatology

Monocytes

MODS

acute pancreatitis

CCR2

Acute pancreatitis (AP) is one of the most common non-malignant gastroenterological disorders leading to hospitalisation of patients¹. In most cases the disease has a mild self-limiting course, but 20% of patients develop severe acute pancreatitis, which is associated with systemic complications. Infected necrosis and/or persistent organ failure are the most perilous complications leading to significant morbidity and mortality^1,2. AP can rapidly progress towards moderately severe or severe AP (SAP), significantly increasing the length of in-hospital stays and ultimately leading to significant healthcare expenditures ³. Until today there is no established treatments for AP or SAP.

According to the revised Atlanta classification, the severe form of acute pancreatitis is defined by the occurrence of persistent organ failure or the presence of a systemic inflammatory response syndrome (SIRS) for longer than 48h⁴. Pancreatitis-induced SIRS is the suggested trigger mechanism for organ damage, affecting mainly the pulmonary, the renal or the cardiovascular system². While SIRS is held responsible for early organ damage like acute respiratory distress syndrome (ARDS) or acute kidney injury (AKI)^2,5, the compensatory anti-inflammatory response syndrome (CARS) is believed to control infection of pancreatic necrosis and late organ failure. Schepers et al. recently showed a lack of correlation between mortality and the onset of early organ failure. This study also challenged the classical view of a biphasic or consecutive SIRS/CARS disease pattern. Beside the clinical observations also animal models of SAP suggest a parallel course of SIRS and CARS⁶, which could explain the observations by Schepers and co-workers⁷. A robust release of pro-inflammatory cytokines and chemokines can induce severe systemic inflammation, leading to multi-organ dysfunction syndrome (MODS)⁵. In parallel, the adaptive immune system counteracts the developing cytokine storm by activating a suppressive immune response^6,8,9, as has been shown for sepsis^10,11. Pancreatitis-related organ complications frequently include the respiratory system^2,7. IL-6 trans-signalling¹⁰ or MCP-1-mediated monocyte/granulocyte mobilisation¹¹ are associated with lung damage during AP. However, the precise pathomechanism behind pancreatitis-induced ARDS is not well understood, one reason being the lack of reproducible SAP animal models

Our aim for the present study was to investigate the cause of secondary organ damage/MODS in a mouse model of SAP induced by partial pancreatic duct ligation and hyperstimulation. In this model the mice developed the full spectrum of disease characteristics of SAP in humans^12,13, including pancreatic necrosis and prominent damage of the respiratory and renal systems. Also, a parallel course of SIRS and CARS have been observed ⁶. We used specific antibody treatment to analyse whether the depletion of neutrophils or monocytes affected the local and systemic immune responses, and how this might impact on the development of organ complications. Our results indicate a general disease mechanism via microcapillary occlusion from enlarged monocytes for the damage of secondary organs during severe inflammatory reactions, such as pancreatitis or sepsis, which offers a therapeutic option for the treatment of MODS.

Disease severity correlates with serum monocyte attracting chemokines

304 pancreatitis patients that had been admitted to our university hospital between 2007 and 2020, were retrospectively classified according to the revised Atlanta criteria into mild, moderate, and severe pancreatitis. SAP patients frequently developed AKI and respiratory failure but also cardiovascular complications were observed (Fig. 1a). At the day of hospital admission leukocyte counts (Gpt/L) as well as C-reactive protein (CRP) were significantly increased in these patients. In addition, serum creatinine, indicating kidney dysfunction, and serum lactate, indicating organ hypoperfusion as a sign of critical illness, were only significantly increased in the group of patients who developed SAP (Fig. 1b). Severe disease was also reflected by a significantly prolonged duration of hospitalisation (Fig. 1c). We next measured the concentration of serum chemokines using Cytometric bead arrays. Monocyte/macrophage attracting chemokines like MCP-1 (CCL-2), MIG (CXCL-9) or IP-10 (CXCL-10) were all significantly increased in SAP patients. Also neutrophil attracting chemokine IL-8 (CXCL-8) was elevated in a severity dependent manner (Fig. 1d). Anaphylatoxins, the cleavage products of complement proteins, are also known to have chemotactic characteristics, but we found no differences for C3a, C4a and C5a with respect to disease severity (Fig. 1e). The correlation between selected chemokines and organ dysfunction was analysed by a correlation analysis of serum lactate and creatinine. Serum lactate showed a positive correlation with MCP-1, IL-8, MIG and IP-10 (Fig. 1f), whereas serum creatinine was significantly correlated with IP-10 and MIG (Fig. 1g). Blood leukocytes counts were positively correlated with serum lactate and creatinine levels, whereas CRP in serum showed only a weak correlation with creatinine but none of the chemokines (supplementary Fig. 1a-c). SAP is therefore associated with a significant mobilization of leukocytes into the vascular system, which may play an important role in the induction of secondary organ damage.

Organ damage in a mouse model of severe acute pancreatitis

In the following experiments, we evaluated AP-associated organ damage in a severe pancreatitis animal model with high similarity to the human situation. Severe acute pancreatitis was induced by partial duct ligation with an additional hyperstimulation of caerulein (50g/kg/bodyweight) in C57Bl/6 mice, as previously described^6,12,13. H&E staining of pancreas sections confirmed the development of a severe necrotizing pancreatitis 3d after the ligation of the duct (Fig. 2a). This was paralleled by a significant increase in serum amylase and lipase activities, which are established markers of disease severity (Fig. 2b). Secondary organ damage was detected by a significant increase of myeloperoxidase activity in lung tissue, which is a consequence of infiltrating leucocytes (Fig. 2c). Histologic analysis of lung sections also showed a pronounced infiltration of leucocytes 3d after the onset of pancreatitis, associated with increased alveolar wall thickness. Beside the infiltration of immune cells, oedema formation (black arrow) is a clear sign of acute lung injury (Fig. 2d). A histological examination of the lung volume by quantification of tissue area against alveolar space, showed a ~ 20% reduction 3d after induction of AP (Fig. 2e). Furthermore, H&E staining of kidney sections showed tubular dilatation (marked by rhomb) and intratubular deposits (marked by arrows), which are both histological signs of AKI and were present in tissue of mice 3d after the onset of pancreatitis, but completely absent in healthy tissue (Fig. 2f)¹⁴. Urinary accumulation of cystatin C, a marker of AKI¹⁵, was detected by dot blot analysis (Fig. 2g) in 3 out of 8 urine samples of pancreatitis animals, but not in control animals. Another indicator of kidney damage was the significantly increased proliferation of tubule cells shown by Ki67 labelling (Fig. 2h). Another indicator of AKI was the detection of a significantly increased expression of neutrophil gelatinase associated lipocalin (NGAL)¹⁶ 3d after the onset of severe acute pancreatitis (Fig. 2h). In contrast to the rather mild pancreatitis model of hormonal (caerulein) hyperstimulation, the partial duct ligation in mice is associated with organ damage such as AKI and respiratory dysfunction, similar to the human situation.

SAP-induced organ dysfunction is associated with infiltration of monocytes, but not neutrophils

Hyperinflammation is believed to play a role in the induction of organ damage. We isolated leukocytes from lung tissue of SAP mice with respiratory dysfunction and evaluated infiltrating leukocytes by flow cytometry. CD4 + T-cells could only be detected in negligible quantities and we therefor analysed cells of the innate immune system, including mature neutrophils (Ly6g+/CD11b-), granulocytic myeloid derived suppressor cells gMDSCs (Ly6g+/CD11b+) and monocytes/macrophages (Ly6g-/CD11b+) by flow cytometry (Fig. 3a). While the number of neutrophils decreased 3d after onset of pancreatitis, gMDSCs and monocytes/macrophages were significantly increased in lung tissue (Fig. 3b). Immunofluorescent labelling confirmed the presence of high numbers of CD11b + cells in lung tissue whereas only few Ly6g + cells could be detected (Fig. 3c). We also labelled CD11b + cells in tissue sections of lung and kidney, which confirmed that macrophages/monocytes are the major cell population infiltrating these organs. At 3d after the onset of pancreatitis, CD11 + cells are visible within the alveolar septs of lung and the glomeruli of the kidney (Fig. 3d and 3e).

Leukocyte depletion ameliorates pancreatitis in mice

In further experiments we analysed the specific role of leukocytes in the development of SAP. Therefore we depleted either neutrophils with an anti Ly6g antibody¹⁷ or monocytes via anti CCR2 antibody¹⁸ 1d before and 1d after onset of pancreatitis. Control mice were left untreated or received rat IgG2 Isotype antibody. The efficacy of the antibody-mediated depletion was confirmed by flow cytometry analysis of splenocytes (Fig. 4a). Treatment with anti-Ly6g antibody resulted in a total loss of Ly6g + neutrophils as well as of Ly6g+/CD11b + gMDSCs, whereas the treatment with anti-CCR2 antibody showed a significant reduction of CD11b + cells, but did not affect the population of Ly6g+/CD11b + gMDSCs (Fig. 4b-d). The effect of anti-Ly6g and anti-CCR2 treatment on pancreatic damage was investigated by histological evaluation. H&E staining confirmed that the induction of pancreatic tissue damage was comparable in all animals, independent off leukocyte depletion (Fig. 4e). Labelling of neutrophils by anti-Ly6g antibodies revealed invasion of only few neutrophils in controls, but a complete absence of neutrophilic granulocytes after depletion with anti-Ly6g antibody in pancreatic tissue (Fig. 4e). Surprisingly, the depletion of monocytes by anti-CCR2 antibody did not influence the large number of CD68 + macrophages in the damaged pancreas (Fig. 4e). Quantitative analysis of neutrophil/macrophage counts confirmed a significant impact of the anti-Ly6g treatment but not of the anti-CCR2 treatment (Fig. 4f). Serum amylase showed a significant reduction under both treatment conditions compared to the isotype antibody control group, whereas serum lipase was reduced in anti-Ly6g treated animals only (Fig. 4g).

Macrophage-dependent pro-inflammatory response

The systemic depletion of monocytes was not associated with a fast reduction in pancreatic macrophages, therefore pancreatic damage appeared less affected. In fact, only a small number of pancreatic macrophageswas positive for CCR2 and thereby sensitive to anti-CCR2 treatment. CCR2-negative monocytes apparently started to proliferate (supplementary Fig. 2a and 2b). Macrophages orchestrate the activation of the adaptive immune system^6,12, so it was not surprising that T-cell activation, as measured by the expression of CD25, CD69 or CD25+/Foxp3 + T_reg cells, was not affected under treatment with anti-Ly6g or anti-CCR2 antibodies. Still, CD8α + cytotoxic T-cells were reduced after monocyte depletion (supplementary Fig. 2c). Pancreatic macrophages, but not infiltrating neutrophils, control the T-cell response via their cytokine profile^6,12. The fact that tissue resident macrophages and not monocyte derived macrophages made up the majority of macrophages explains the missing effect of CCR2 depletion on T cell activation. Analysis of serum cytokines gave additional evidence that CCR2 depletion did not affect the systemic pro-inflammatory response (supplementary Fig. 3).

Depletion of Monocytes ameliorates pancreatitis-induced organ damage

In a next step we investigated the influence of neutrophil and monocyte depletion on pancreatitis-induced organ damage. We analysed the composition of leukocytes that had been isolated from lung tissue of mice by flow cytometry. Independent of the treatment with depleting antibodies we observed a dramatic reduction of Ly6g+/CD11b- mature neutrophils 3d after induction of pancreatitis (Fig. 5a). Neutrophil depletion by anti-Ly6g antibody resulted in no further reduction of Ly6g+/CD11b- neutrophils, but in a significant reduction of Ly6+/CD11b + gMDSCs. The depletion via anti-CCR2 antibody significantly decreased CD11b+/Ly6g- monocytes whereas the population of Ly6g+/CD11b + gMDSCs was not affected by the treatment (Fig. 5a and 5b). H&E staining of lung tissue showed no significant differences between the isotype control group and both treatment groups (Fig. 5c). However, labelling of CD11b revealed a dramatic increase of CD11b + cells (gMDSC/monocytes) in isotype and anti-Ly6g treated mice but only a minor increase in mice which received the anit-CCR2 antibody (Fig. 5c and 5d). Myeloperoxidase is expressed in large amounts in monocytes and neutrophils¹⁹ and as such a reliable marker of their tissue invasion. Measurements in lung tissue homogenate confirmed pancreatitis-induced elevated myeloperoxidase activities, which were significantly reduced in both antibody treatment groups compared to the isotype controls (Fig. 5e), To evaluate the respiratory function/dysfunction of these mice we analysed arterial blood samples and found in all animals suffering from SAP a significant reduction in oxygen saturation and increased serum lactate levels (Fig. 5f-g). Pancreatitis animals in general had decreased pO₂ and glucose levels, whereas pCO₂ and ctHb were elevated (supplementary Fig. 4a). Serum electrolytes remained unchanged (supplementary Fig. 4b). While the depletion of neutrophils had no beneficial effect on respiratory function, the monocyte depletion significantly increased oxygen saturation and reduced serum lactate levels (Fig. 5f-g). Serum creatinine levels were measured as indicator of AKI in mice. Mice which received isotype control or anti-Ly6g antibody showed a dramatic increase of serum creatinine levels, whereas creatinine levels from mice treated with anti-CCR2 antibody were comparable to untreated control mice (Fig. 5h). Immunohistochemical labelling of CD11b identified leukocytes in kidney glomeruli of control or anti-Ly6g treated, but not of anti-CCR2 treated animals (Fig. 5i).

SAP mice have Increased numbers of enlarged blood monocytes

Next, we analysed whether the identified CD11b + cells in histologic sections of lung and kidney were resident tissue cells, or whether these monocytes are circulating in the vascular system. Flow cytometric analysis of blood samples from SAP mice showed a significant increase of Ly6g + neutrophils and of CD11b + monocytes (Fig. 6a and b), which is in line with our observations in patients (Fig. 1). In contrast to the boosted response of the innate immune system, CD4 + T-helper cells were significantly reduced. (Fig. 6b). Monocytes and neutrophils, are both mobilized from bone marrow and associate with a more severe course of disease^12,17,20. After induction of pancreatitis only a minority of circulating cells are Ly6g+/CD11b- mature neutrophils, whereas double positive Ly6g+/CD11b+ (gMDSCs) as well as Ly6g-/CD11b + monocytes were significantly increased, similar to our previous findings in spleen and lung tissue (Fig. 6c). As the monocyte depletion in our mouse model did not alter the local and systemic immune responses we speculated that a cell-mediated effect might be operative in the induction of organ damage. Beside their signalling involvement in immune regulation networks, the mechanical characteristics of myeloid derived cells have been discussed in a pathophysiological context in the recent literature²¹. In further experiments we isolated blood cells from mice and analysed their mechanical properties by real-time fluorescence and deformability cytometry (RT-FDC) after gating for 1) Ly6g+/CD11b- neutrophils, 2) Ly6g+/CD11b + gMDSCs and 3) Ly6g-/CD11b + monocytes (Fig. 6d)²². Deformation and cell size were analysed in Ly6g and CD11b labelled populations indicating a general increase in cell size (Fig. 6e). A comparison of six experimental replicates demonstrates a significant increase in cell size after 3d of all investigated populations (Fig. 6f). In contrast, Ly6g + neutrophils as well as gMDSCs showed an increased deformation at 0d compared to CD11b+/Ly6g- monocytes (Fig. 6g). In fact, deformation of monocytes increased after onset of pancreatitis but did not reach the level of neutrophiles or gMDSCs. Applying an analytical model to our data enables to calculate the elastic modulus as an intrinsic material property²³. The results indicate a significant increase for neutrophiles and gMDSCs 3d after onset of pancreatitis, whereas the elasticity of monocytes was slightly, but not significantly smaller. During pancreatitis monocytes are mobilized in high numbers from the bone marrow and subsequently they circulate in the blood (Fig. 6b). The size of these monocytes is increased (Fig. 6f) but their flexibility is not increased accordingly (Fig. 6h). We hypothesized that these monocytes accumulate in the lung vascular system and obstruct the microcirculation, which results in respiratory dysfunction. In the next step, we therefore isolated monocytes from lung tissue and analysed them by RT-FDC^22,24. After the induction of pancreatitis we observed a significant increase of cell size and deformation of monocytes in lung tissue, similar to our previous observation in whole blood (Fig. 7a-b). In to the same extent like monocytes isolated from blood, we found no significant differences in the elastic modulus (Fig. 7c). These findings associate pancreatitis with increasing monocyte size but maintained elastic modlus, leading to a congestion of monocytes in small vessels that causes organ damage. Immunofluorescent labelling of CD11b, Ly6g and VE-Cadherin supported this hypothesis. Increased numbers of CD11b + cells were observed within the vascular system of lung after onset of pancreatitis (Fig. 7d). CCR2 + monocytes accumulate within the tissue, whereas CD68 + resident alveolar macrophages are not increased (supplementary Fig. 5a). A similar picture we previously saw within the glomeruli of kidney in animals with SAP (supplementary Fig. 5b).

The induction of a multi organ dysfunction syndrome during a sever course of acute pancreatitis⁴ is associated with dramatically increased morbidity and mortality and increasing health care utilization³. The underlying pathomechanisms behind pancreatitis associated MODS are unknown, and a causal therapy is missing. In our study we investigated organ damage in a pancreatitis mouse model which develops SIRS^6,12 as well as MODS, both indicating severe acute pancreatitis according to the revised Atlanta classification⁴. Acute pancreatitis is associated with (mainly) necrosis of acinar cells, thus leading to local tissue damage and the initiation of an overwhelming pro-inflammatory immune response. Apparently, the extent of local damage has a critical influence on the strength of the inflammatory response and is therefore suggested to play an essential role in the induction of secondary organ damage and systemic complications^2,5,10. It`s the overwhelming immune response of acute pancreatitis which defines the severity of the disease course. An activation of the transcription factor NFκB in acinar cells occurs in parallel with intra acinar protease activation, and is an early event in disease manifestations^25,26. The release of cytokines and chemokines then recruits immune cells to the pancreas where they contribute to the local damage^{12,17,20,27,28} and enhance the immune response^6,29,30. Previous experiments suggest that SIRS and CARS in mouse models of SAP are not subsequent events, but occur in parallel^6,8. Also, recent data from patients indicate a severity-dependent anti-inflammatory response in the presence of SIRS^7,9. The activated immune response in the course of SAP is similar to other severe inflammatory responses such as sepsis³¹ or sever burn traumata³² which all may comprise the induction of MODS^33,34. This suggests a common inflammatory response mechanism leading to secondary organ dysfunction. The most affected organs are the renal and the respiratory system which have one thing in common, their vascular system of micro-capillaries. A disturbed microcirculation therefore could contribute to organ failure³³. We observed a significant increase of two cell populations of the innate immune system in the blood after the onset of pancreatitis: 1.) CD11b+/Ly6g- monocytes which could effectively be depleted by the usage of anti-CCR2 antibody¹⁸, and 2.) Ly6g+/CD11b + cells which represent gMDSCs³⁵ and could successfully be depleted by anti-Ly6g antibody application. The pre-treatment with anti-Ly6g depleting antibody results in a complete loss of gMDSCs and Ly6g + neutrophils and attenuates the local pancreatic damage but has no effect on disease related organ dysfunction. It is well known that neutrophils contribute to acinar cell damage via the release of reactive oxygen²⁰ species or pro-inflammatory cytokines like TNFα¹⁷. Our results confirm these findings but suggest that neutrophils are not involved in the initiation of systemic complications. Moreover, the number of Ly6g+/CD11b- mature neutrophils significantly decreased in lung tissue after the induction of pancreatitis, presumably as a consequence of an induced immunosuppression during SAP. Apparently, the removal of Ly6+/CD11b + gMDSCs and Ly6g+/CD11b- mature neutrophils in mice by depleting antibodies had no demonstrable effect on the systemic immune response because the number of mature neutrophils decreases during the induction of pancreatitis anyway.

In contrast, the depletion of monocytes by anti CCR2 antibody demonstrated that a macrophage-mediated immune response is much more complex. CD11b+/Ly6g- monocytes were depleted from spleen and lung tissue of pancreatitis-induced mice, but the tissue-resident macrophages of the pancreas were not affected. Also, the systemic pro-inflammatory immune response did not differ between anti-CCR2 and isotype control mice, which supports the assumption that resident pancreatic macrophages initiate the development of SIRS/CARS by activating the adaptive immune system⁶. Tissue resident macrophages^36,37 are independent of circulating monocytes. They start to proliferate in response to acinar cell damage and orchestrate the local and systemic immune response. While the depletion of CCR2 + monocytes did neither affect the systemic immune response, nor necrosis clearance by resident pancreatic macrophages, the development of organ dysfunction was significantly reduced. Monocyte–depleted mice had better oxygen saturation and less elevated serum creatinine levels. Monocytes develop from precursor cells in the bone marrow and circulate in the blood³⁸, whereas tissue resident macrophages develop from invading monocytes starting with embryogenesis³⁹. After transmigration into the tissue, they act as monocyte derived macrophages. In the adult, macrophages play a role in almost all inflammatory responses, including wound repair processes and it has been shown in adult guinea pigs that, when macrophage influx is blocked by administration of anti-macrophage serum, wound healing is severely impaired⁴⁰. Following the induction of pancreatitis we saw a massive release of monocytes from bone marrow into the vascular system, but, surprisingly, no rapid transmigration into the site of pancreatic damage. The depletion of these circulating monocytes did not affect the local and systemic pro-inflammatory immune response, but significantly ameliorated organ complications. These findings suggest that the induction of MODS in severe pancreatitis is not mediated by an immunological signalling pathway but related to the physical properties of the monocytes²¹. Lung and kidney are characterised by microvascular systems and the passage of large cells, like monocytes, through these capillary systems requires a flexible adaptation to their small capillaries⁴¹. LPS activated monocytes acquire stiffer physical properties, leading to a prolonged retention in the capillary system of the lung⁴². Here we demonstrate that the induction of pancreatitis also changed the mechanical behaviour of myeloid derived cells dramatically. During pancreatitis, Ly6g + neutrophils, gMDSCs and CD11b + monocytes are all characterized by an increased cell size. However, neutrophils and gMDSCs revealed a high deformation compared to CD11b + monocytes. As a consequence, large numbers of these rather large and stiff monocytes remain circulating in the vascular system while they do not transmigrate into the pancreatic tissue. The depletion of these monocytes, but not of neutrophils and gMDSCs significantly ameliorated lung and kidney damage. A retrospective analysis of clinical data from patients demonstrated a clear correlation of leukocyte count and lactate, as marker for lung damage, or serum creatinine as marker for kidney damage. In addition, the serum level of the Monocyte attracting chemokines MCP-1, which is the ligand of CCR2, was significantly increased in patients with SAP compared to milder forms of the disease. This underlines the importance of monocytes for the disease severity and the induction of MODS in patients with SAP. Severe immune responses like in sepsis or severe trauma are also frequently associated with secondary organ damages like AKI or ARDS. We speculate that our observation is not a pancreatitis specific mechanism. Recent data suggest a parallel occurrence of SIRS and CARS during acute pancreatitis⁶, in a way that a severe pro-inflammation response, is counter-regulated by parts of the adaptive^6,43 and innate immune system⁴⁴ in order to prevent hyperinflammation and systemic shock. Monocytes are released from the bone marrow in response to the pancreatitis-induced pro-inflammation and enter the vascular system. A parallel compensatory systemic immunosuppressive reaction prevents their differentiation and, as a consequence, the cells remain in the vascular system. These large numbers of monocytes, isolated from blood as well as from lung tissue, which are characterized by increased size and unchanged elasticity are especially problematic for organs with small capillary networks like the lungs or the glomeruli of kidney. We propose that these monocytes represent a common pathomechanism and are the reason why severe immune reactions with a parallel SIRS/CARS lead to damaged renal and pulmonary system as it is known for pancreatitis, burn traumata and sepsis^2,4,33,45. Recent genome wide association studies of lung tissue from Covid-19 patients identified an association of severe disease courses with increased CCR2 expression⁴⁶. Monoclonal anti-CCR2 antibody therapy, which is already used for rheumatoid arthritis⁴⁷ may be a treatment option against MODS during severe acute pancreatitis.

In summary, we could demonstrate a massive mobilisation of monocytes from the bone marrow in response to pro-inflammation during SAP. The number of circulating monocytes was dramatically increased after onset of pancreatitis and resulted in an impaired microcirculation in capillary networks. Beside the number of monocytes also their physical properties changed. Here, the cell size is increased without a compensatory increase in elasticity. This accumulation of monocytes in narrow capillaries of the lungs and glomeruli caused organ dysfunction, characterized by AKI and ARDS in a mouse model of SAP. The depletion of monocytes, but not of neutrophils or gMDSCs, was sufficient to significantly ameliorate lung and kidney damage. Monocytes represent a promising therapeutic target for the prevention of organ complications in SAP and possible other inflammation-related disorders affecting microcapillaries.

Animal Model

Wildtype C57Bl/6 mice were purchased from Charles River Laboratories (Sulzfeld, Germany). SAP was induced by partial duct ligation and an additional caerulein injection at day 2 (50mg/kg bodyweight), as described previously^6,8,12,13. To deplete Monocytes and Neutrophils mice were treated with anti-Ly6G antibody¹⁷ (BioXcell, Lebanon USA, BP0075-1), with rat anti-mouse-CCR2 antibody MC-21¹⁸ (a kind gift from Matthias Mack, Regensburg Germany, lot 1480/02) and as control with isotype IgG2a (BioXcell, BP0089) or isotype IgG2b (BioXcell BE0090). Anti-Ly6G antibody and isotype IgG2a antibody were injected i.p. with 200µg in 200µl PBS one day before and the day after duct ligation. Anti-CCR2 antibody and Isotype IgG2b antibody were injected i.p. with 20µg in 200µl PBS at the day of duct ligation and one and two days after surgery. Mice were sacrificed 72 hours after duct ligation and organs were used for analysis. Serum was stored at -80°C. Pancreas, lung and kidney were fixed in 4.5% formaldehyde and embedded in paraffin, embedded in TissueTec or frozen in liquid nitrogen for rtPCR analysis. Spleen and lung were taken for FACS analysis.

Antibodies and Reagents

The following antibodies were used for FACS staining, IHC staining and immunofluorescence staining: Anti-mouse CD4 BV650 (BioLegend 100546), anti-mouse-CD11b PerCP Cy55 (BioLegend, 101228), anti-mouse-Ly6G BV421 (BioLegend, 127628), anti-mouse-Ly6C BV605 (BioLegend 128036), anti-CD25-PECy7 (BioLegend, 102016), anti-CD69 BV510 (BioLegend, 104532), anti-CD8a BV605 (BioLegend, 100743), anti-FoxP3 APC (Miltenyi Biotec, 130-111-601), anti-mouse-CD11b (abcam, Ab133357), anti-mouse-CD68 (antibody-online, ABIN181836), anti-CCR2 (abcam, ab273050) anti-Ki67 (Bethyl, IHC-00375), anti-mouse-CD11b (abcam, Ab133357), anti-mouse-Ly6g (abcam, Ab25377), anti-mouse-Cystatin C (Novus biologicals, NB100-1033), anti-VE-cadherin (abcam, ab7047-50) anti-rabbit-HRP (DAKO, K4003), anti-mouse-HRP (DAKO, K4001). Caerulein was obtained from Sigma Aldricht (C9026-1MG, Munich, Germany), human myeloperoxidase from Calbiochem (Cat# 475911).

Flow cytometry analysis of spleen

To analyse splenocytes, spleen was taken and mashed through a 70µm cell strainer. Cells were washed with PBS and centrifuged at 300g for 6 minutes. To lyse erythrocytes pellet was resuspended in 1 ml lysis buffer for 5 minutes. After washing with PBS and centrifugation at 300g for 6 minutes cells were stained with fluorescence antibodies. Extracellular staining was performed with surface antibodies (1:50): Anti-CD4, anti-CD11b, anti-Ly6G, anti-Ly6C, anti-CD25, anti-CD69, anti-CD8a. To block non-specific Fc mediated interactions cells were pre-treated with anti-mouse CD16/CD32 antibody for 5 min (1:50). Extracellular antibodies were incubated for 30 minutes at 4°C. Cells were washed with PBS and centrifuged at 300g for 6 minutes. Pellet was resuspended in 150µl FACS buffer and measured immediately. Data were analysed with BD FACS DIVA Software and FlowJo.

Flow cytometry analysis of lung

To analyse leucocytes from lung, lungs were removed from mice and were dissociated according to the protocol of lung dissociation kit from Miltenyi (130-095-927, Bergisch Gladbach, germany). Staining for flow cytometry was performed as described in splenocyte staining. Data were analysed with BD FACS DIVA Software and FlowJo.

Serum Amylase and Lipase

Serum amylase and lipase measurement was performed with colorimetric assay from Roche/Hitacho (Amyl Ref 11876473316; Lip Ref 11821792216).

Oxygen saturation of blood

Arterial Blood was taken with a capillary from arteria carotis communis and was measured with BGA analysis machine (ABL90 FLEX, Radiometer GmbH, Germany). Carotis communis was prepared as described previously. Parameters like pO₂, pCO₂, glucose, lactat, etc. were measured and analysed.

MPO activity

MPO measurement was performed like previously described^6,17,48,49. Lung tissue was homogenized on ice in 20mM potassium phosphate buffer (pH 7.4) and centrifuged at 14.000 rcf. The pellet was resuspended in potassium phosphate buffer (pH 6.0) containing 0.5% cetyltrimethylammoniumbromide. Suspension was frozen and thawed in 4 cycles and centrifuged at 14.000 rcf. MPO activity was measured in 50 mM potassium phosphate buffer (pH 6.0) containing 0.53 mM O-dianisidine and 0.15 mM H₂O₂. The MPO activity was measured as kinetic over time with a Spectramax Spectrophotometer (Molecular devices). Purified human myeloperoxidase was used as standard, the final MPO activity was calculated against protein content of the samples.

Histology, Immunohistochemistry and Immunfluorescence staining

For H&E staining the lung, pancreas and kidney were embedded in paraffin, 3µm thick slides were cut by microtome. Slides were deparaffined in Xylol, and hydrated in methanol followed by decreased ethanol concentrations, hematoxylin and eosin staining was performed as described previously^13,17. For lung analysis slides were scanned with slide scanner (Pannoramic MDI BF/FL; Sysmex, Norderstedt, Germany) and analysis were made with Software Pattern Quant from Sysmex Quant Center.

For immunohistochemistry staining slides were dewaxed and cooked in antigenretrieval solution (Dako target retrieval solution, ref. S1699). Rabbit anti-mouse-CD11b antibody (abcam, cambride UK, Ab133357) was used in a concentration of 1:400 over night at 4°C. Anti-rabbit antibody was incubated for 1 hour at RT. DAB Kit (Vector, Burlingame USA, SK4100) was used to visualize CD11b positive cells.

For immunofluorescence staining TissueTec embedded slides were used. Rabbit-anti-mouse-CD11b, Ly6g, CD68, CCR2 or VE-cadherin antibody was used as 1:400. Secondary antibody was used in a concentration of 1:200. DAPI was used for nucleus staining. All slides were scanned with Pannoramic slide scanner and analysis were made with Sysmex Quant Center Nucleus or Pattern.

Cytokines in Serum

The chemokines MCP-1, MIG, IP-10, IL8 and complement system anaphylatoxins C3a, C4a and C5a were measured in human serum from patients with severe, moderate and mild form of acute pancreatitis. Measurements were performed with CBA Kits from BD (Human Anaphylatoxin Kit 561418BD; Human Chemokine Kit 552990; BD San Jose, USA). Murine chemokines and cytokines were measured by BD cytometric bead array (CBA) mouse inflammation kit in serum of mice.

Real Time Fluorescence and Deformability Cytometry

Mechanical characterization of monocytes was performed in whole blood and lung monocytes isolated from pancreatic duct-ligated mice as well as untreated controls, respectively. For monocyte identification in whole blood, cells were labeled with Ly6g (APC conjugated) and CD11b (FITC conjugated) fluorescent antibodies. Lung monocytes were isolated (lung dissociation Kit, Miltenyi) by usage of monocytes isolation kit (EasySep mouse Monocyte Isolation Kit, Stemcell, 19861A). After isolation lung monocytes are resuspended in PBS (without Ca²⁺ and Mg²⁺) complemented with 0.6% (w/v) Methylcellulose to a concentration of approximately 10 x 10⁶ cells per ml.

High-throughput mechanical analysis was done by real-time fluorescence and deformability cytometry using the AcCellerator system with fluorescence extension (Zellmechanik Dresden GmbH)^22,24. Briefly, the system consists of an inverted microscope, where a microfluidic chip with a 300 µm long constriction is assembled on a xy-stage. We use a channel cross-section of 20 µm x 20 µm for whole blood measurements and a 15 µm x 15 µm cross-section for lung monocytes. In all measurements a flow rate of 0.06 µl/s is applied. For each condition data from several thousand monocytes has been obtained. Only cells are analysed with an area ratio < 1.05 (ratio between actual cell area and cell area detected image analysis) to ensure a full representation of cell shape.

Derivation of elastic modulus was done using an analytical model published earlier²³. Here, the flow profile around a moving cell is calculated assuming Stokes flow and deformation is predicted from coupling the resulting stress-distribution to the cell surface applying linear elasticity theory and assuming the cell being a homogeneous material.

Data were analysed with the Software ShapeOut.

qrt-PCR

RNA preparation was performed with Trizol as described before¹². qrt-PCR was performed for NGal (5´-CACCACGGACTACAACCAGTTCGC-3´; 5´-TCAGTTGTCAATGCATTGGTCGGTG-3´). As house-keeping gene 5S (5´-GCCCGATCTCGTCTGATCTC-3´; 5´-GCCTATCAGCACCCGGTATTC-3´) was analysed and markers were normalized to 5S.

Statistical analysis

The statistical evaluation as well as the graphical presentation of the experiments were carried out in GraphPad Prism and SigmaPlot. All graphs were generated with the standard error of the mean (SE). As significance test the two-sided students T-test for unpaired samples was used, all samples p > 0.05 were classified as significant. Patients samples were analysed by Kruskal-Wallis test followed by Dunn’s multiple comparison test, significant differences were defined by p < 0.05, the correlation was tested by spearman rank order correlation. A statistical significant differences for more than 3 groups 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. Statistical analysis of RT-FDC data has been performed using linear mixed models, where we compared two groups of experimental replicates, respectively⁵⁰. While fixed effects account for the actual effect, i.e., increase or decrease in deformation, cell size or elastic modulus, random effects represent systematic or random measurement bias.

Acknowledgments:

Specific author contributions: Concept of the study MS, FUW, AW. Data acquisition and interpretation: MS, AW, JG, AAA, DB, OO, MN, MM, SR, BMB, SFvB. Writing committee MS, FUW, AAA, MML. Correction of manuscript and approval of final version: all.

Funding: This work was supported by Deutsche Forschungsgemeinschaft (DFG SE 2702/2-1, AG 203/4-1 and GRK 1947), the PePPP center of excellence MV (ESF/14-BM-A55-0045/16) and the EnErGie/P2 Project (ESF/14-BM-A55-0008/18), the Bundesministerium für Bildung und Forschung (ZIK HIKE grant to OO under grant agreement 03Z22CN11) and the Deutsches Zentrum für Herz-Kreislauf-Forschung (Postdoc startup grant to OO under grant agreement 81X3400107).

Conflict-of-Interest

OO is co-founder of Zellmechanik Dresden GmbH distributing the technology of real-time deformability cytometry.

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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.

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).
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Obstruction of Capillaries by circulating large Monocytes causes Multi Organ Dysfunction Syndrome (MODS) in Acute Pancreatitis

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