Image_1_Complement Activation via the Lectin and Alternative Pathway in Patients With Severe COVID-19.jpg

Complement plays an important role in the direct defense to pathogens, but can also activate immune cells and the release of pro-inflammatory cytokines. However, in critically ill patients with COVID-19 the immune system is inadequately activated leading to severe acute respiratory syndrome (SARS) and acute kidney injury, which is associated with higher mortality. Therefore, we characterized local complement deposition as a sign of activation in both lungs and kidneys from patients with severe COVID-19. Using immunohistochemistry we investigated deposition of complement factors C1q, MASP-2, factor D (CFD), C3c, C3d and C5b-9 as well as myeloperoxidase (MPO) positive neutrophils and SARS-CoV-2 virus particles in lungs and kidneys from 38 patients who died from COVID-19. In addition, tissue damage was analyzed using semi-quantitative scores followed by correlation with complement deposition. Autopsy material from non-COVID patients who died from cardiovascular causes, cerebral hemorrhage and pulmonary embolism served as control (n=8). Lung injury in samples from COVID-19 patients was significantly more pronounced compared to controls with formation of hyaline membranes, thrombi and edema. In addition, in the kidney tubular injury was higher in these patients and correlated with lung injury (r=0.361*). In autopsy samples SARS-CoV-2 spike protein was detected in 22% of the lungs of COVID-19 patients but was lacking in kidneys. Complement activation was significantly stronger in lung samples from patients with COVID-19 via the lectin and alternative pathway as indicated by deposition of MASP-2, CFD, C3d and C5b9. Deposits in the lung were predominantly detected along the alveolar septa, the hyaline membranes and in the alveolar lumina. In the kidney, complement was significantly more deposited in patients with COVID-19 in peritubular capillaries and tubular basement membranes. Renal COVID-19-induced complement activation occurred via the lectin pathway, while activation of the alternative pathway was similar in both groups. Furthermore, MPO-positive neutrophils were found in significantly higher numbers in lungs and kidneys of COVID-19 patients and correlated with local MASP-2 deposition. In conclusion, in patients who died from SARS-CoV-2 infection complement was activated in both lungs and kidneys indicating that complement might be involved in systemic worsening of the inflammatory response. Complement inhibition might thus be a promising treatment option to prevent deregulated activation and subsequent collateral tissue injury in COVID-19.</p

Image_3_Complement Activation via the Lectin and Alternative Pathway in Patients With Severe COVID-19.jpeg

Complement plays an important role in the direct defense to pathogens, but can also activate immune cells and the release of pro-inflammatory cytokines. However, in critically ill patients with COVID-19 the immune system is inadequately activated leading to severe acute respiratory syndrome (SARS) and acute kidney injury, which is associated with higher mortality. Therefore, we characterized local complement deposition as a sign of activation in both lungs and kidneys from patients with severe COVID-19. Using immunohistochemistry we investigated deposition of complement factors C1q, MASP-2, factor D (CFD), C3c, C3d and C5b-9 as well as myeloperoxidase (MPO) positive neutrophils and SARS-CoV-2 virus particles in lungs and kidneys from 38 patients who died from COVID-19. In addition, tissue damage was analyzed using semi-quantitative scores followed by correlation with complement deposition. Autopsy material from non-COVID patients who died from cardiovascular causes, cerebral hemorrhage and pulmonary embolism served as control (n=8). Lung injury in samples from COVID-19 patients was significantly more pronounced compared to controls with formation of hyaline membranes, thrombi and edema. In addition, in the kidney tubular injury was higher in these patients and correlated with lung injury (r=0.361*). In autopsy samples SARS-CoV-2 spike protein was detected in 22% of the lungs of COVID-19 patients but was lacking in kidneys. Complement activation was significantly stronger in lung samples from patients with COVID-19 via the lectin and alternative pathway as indicated by deposition of MASP-2, CFD, C3d and C5b9. Deposits in the lung were predominantly detected along the alveolar septa, the hyaline membranes and in the alveolar lumina. In the kidney, complement was significantly more deposited in patients with COVID-19 in peritubular capillaries and tubular basement membranes. Renal COVID-19-induced complement activation occurred via the lectin pathway, while activation of the alternative pathway was similar in both groups. Furthermore, MPO-positive neutrophils were found in significantly higher numbers in lungs and kidneys of COVID-19 patients and correlated with local MASP-2 deposition. In conclusion, in patients who died from SARS-CoV-2 infection complement was activated in both lungs and kidneys indicating that complement might be involved in systemic worsening of the inflammatory response. Complement inhibition might thus be a promising treatment option to prevent deregulated activation and subsequent collateral tissue injury in COVID-19.</p

Table_1_Complement Activation via the Lectin and Alternative Pathway in Patients With Severe COVID-19.xlsx

Complement plays an important role in the direct defense to pathogens, but can also activate immune cells and the release of pro-inflammatory cytokines. However, in critically ill patients with COVID-19 the immune system is inadequately activated leading to severe acute respiratory syndrome (SARS) and acute kidney injury, which is associated with higher mortality. Therefore, we characterized local complement deposition as a sign of activation in both lungs and kidneys from patients with severe COVID-19. Using immunohistochemistry we investigated deposition of complement factors C1q, MASP-2, factor D (CFD), C3c, C3d and C5b-9 as well as myeloperoxidase (MPO) positive neutrophils and SARS-CoV-2 virus particles in lungs and kidneys from 38 patients who died from COVID-19. In addition, tissue damage was analyzed using semi-quantitative scores followed by correlation with complement deposition. Autopsy material from non-COVID patients who died from cardiovascular causes, cerebral hemorrhage and pulmonary embolism served as control (n=8). Lung injury in samples from COVID-19 patients was significantly more pronounced compared to controls with formation of hyaline membranes, thrombi and edema. In addition, in the kidney tubular injury was higher in these patients and correlated with lung injury (r=0.361*). In autopsy samples SARS-CoV-2 spike protein was detected in 22% of the lungs of COVID-19 patients but was lacking in kidneys. Complement activation was significantly stronger in lung samples from patients with COVID-19 via the lectin and alternative pathway as indicated by deposition of MASP-2, CFD, C3d and C5b9. Deposits in the lung were predominantly detected along the alveolar septa, the hyaline membranes and in the alveolar lumina. In the kidney, complement was significantly more deposited in patients with COVID-19 in peritubular capillaries and tubular basement membranes. Renal COVID-19-induced complement activation occurred via the lectin pathway, while activation of the alternative pathway was similar in both groups. Furthermore, MPO-positive neutrophils were found in significantly higher numbers in lungs and kidneys of COVID-19 patients and correlated with local MASP-2 deposition. In conclusion, in patients who died from SARS-CoV-2 infection complement was activated in both lungs and kidneys indicating that complement might be involved in systemic worsening of the inflammatory response. Complement inhibition might thus be a promising treatment option to prevent deregulated activation and subsequent collateral tissue injury in COVID-19.</p

Clinico-pathologic characteristics of analyzed samples.

<p>Abbreviations: HPP: hyperplastic polyp; SSA/P: sessile serrated polyp/adenoma; TSA: traditional serrated adenoma; TbA: tubular adenoma; Ca: invasive colorectal carcinoma; Met: Metastasis; BRAF c600: B1 Rapidly accelerated fibrosarcoma codon 600 mutation; KRAS c12/13: Kirsten rat sarcoma codon 12 or 13 mutation.; n.t.: not tested; -: not applicable.</p

Abi1 expression analysis in specimens and cell lysates. A

<p>, Distribution of Abi1 expression in healthy and inflamed mucosa, hyperplastic polyps (HPP), sessile serrated polyps/adenomas (SSA/P), traditional serrated adenomas (TSA), tubular adenomas (TbA), invasive colorectal carcinoma (Ca) and metastases (Met). All values except BRAF-mutated TbA and carcinoma (each n = 1) are shown in box and whisker plot. Green squares represent maximum outliers, red squares represent minimum outliers. For inflamed mucosa, median, 1^st and 3^rd quartile are equal (score = 4). <b>B</b>, Statistical differences in Abi1 expression among all examined tissue specimens with respect to mutation status and, where applicable, microsatellite stability of each lesion. The lane for KRAS-mutated HPP is highlighted with a yellow background, the lane for KRAS-mutated invasive carcinoma is highlighted with a red background. The undermost line shows the number of examined samples in each group. <i>M: healthy mucosa; IM: inflamed mucosa; HP wt, HP K, HP B: wild-type, KRAS-mutated and BRAF-mutated hyperplastic polyps; SP wt, SP K, SP B: wild-type, KRAS-mutated and BRAF-mutated sessile serrated polyps/adenomas; TA wt, TA K: wild-type and KRAS-mutated traditional serrated adenomas; TbA wt, TbA K: wild-type and KRAS-mutated tubular adenomas; CA wt, CA K, CA MI: wild-type, KRAS-mutated and microsatellite-instable carcinomas; Met wt, Met K: wild-type and KRAS-mutated metastases; n.s.: not significant; *p<0.1;** p<0.05;*** p<0.01.</i></p

Proposed model for the regulation of actin dynamics via KRAS, PI3K and Abi1.

<p>Ligand-binding to membrane-associated receptor tyrosine kinase (RTK) leads to an activation of RAS, which is constitutively activated in mutant KRAS (*). Activated KRAS activates - among others - both the B1 Rapidly accelerated fibrosarcoma (BRAF)- and Phosphatidylinositol-3-kinase (PI3K)-pathway, only the latter leading to activation of the Abi1/Sos1/Eps8 complex. Via activation of the small GTPase Rac, this leads to reorganization of the actin cytoskeleton and to a change in cellular shape. <i>RTK: receptor tyrosine kinase; KRAS: Kirsten rat sarcoma; BRAF: B1 Rapidly accelerated fibrosarcoma</i>; <i>PI3K: Phosphatidylinositol-3-kinase; Abi1: Abelson interactor 1; Eps8: Epidermal growth factor receptor kinase substrate</i>; <i>Sos1: Son of sevenless homolog 1</i>; <i>Rac: Ras-related C3 botulinum toxin substrate.</i></p

Expression of Abelson Interactor 1 (Abi1) Correlates with Inflammation, KRAS Mutation and Adenomatous Change during Colonic Carcinogenesis

<div><h3>Background</h3><p>Abelson interactor 1 (Abi1) is an important regulator of actin dynamics during cytoskeletal reorganization. In this study, our aim was to investigate the expression of Abi1 in colonic mucosa with and without inflammation, colonic polyps, colorectal carcinomas (CRC) and metastases as well as in CRC cell lines with respect to BRAF/KRAS mutation status and to find out whether introduction of KRAS mutation or stimulation with TNFalpha enhances Abi1 protein expression in CRC cells.</p> <h3>Methodology/Principal Findings</h3><p>We immunohistochemically analyzed Abi1 protein expression in 126 tissue specimens from 95 patients and in 5 colorectal carcinoma cell lines with different mutation status by western immunoblotting. We found that Abi1 expression correlated positively with KRAS, but not BRAF mutation status in the examined tissue samples. Furthermore, Abi1 is overexpressed in inflammatory mucosa, sessile serrated polyps and adenomas, tubular adenomas, invasive CRC and CRC metastasis when compared to healthy mucosa and BRAF-mutated as well as KRAS wild-type hyperplastic polyps. Abi1 expression in carcinoma was independent of microsatellite stability of the tumor. Abi1 protein expression correlated with KRAS mutation in the analyzed CRC cell lines, and upregulation of Abi1 could be induced by TNFalpha treatment as well as transfection of wild-type CRC cells with mutant KRAS. The overexpression of Abi1 could be abolished by treatment with the PI3K-inhibitor Wortmannin after KRAS transfection.</p> <h3>Conclusions/Significance</h3><p>Our results support a role for Abi1 as a downstream target of inflammatory response and adenomatous change as well as oncogenic KRAS mutation via PI3K, but not BRAF activation. Furthermore, they highlight a possible role for Abi1 as a marker for early KRAS mutation in hyperplastic polyps. Since the protein is a key player in actin dynamics, our data encourages further studies concerning the exact role of Abi1 in actin reorganization upon enhanced KRAS/PI3K signalling during colonic tumorigenesis.</p> </div

Abi1 expression in analyzed samples.

<p>All values shown as mean ± SD; abbreviations: HPP: hyperplastic polyp; SSA/P: sessile serrated polyp/adenoma; TSA: traditional serrated adenoma; TbA: tubular adenoma; Ca: invasive colorectal carcinoma; Met: Metastasis; BRAF c600: B1 Rapidly accelerated fibrosarcoma codon 600 mutation; KRAS c12/13: Kirsten rat sarcoma codon 12/13 mutationMSI: microsatellite instable tumors; n: number of examined samples; n.a.: not applicable due to low sample number.</p

Abi1 in colorectal cancer cell lines. A

<p>, Abi1 immunoblotting of colorectal carcinoma whole cell line lysates with different KRAS/BRAF mutation status shows upregulation of Abi1 in KRAS-mutated SW620 and SW1116, but only a faint signal in BRAF-mutated Colo205 cells. <b>B</b>, Immunoblotting of CHD-1 and HDC-9 cell lysates show overexpression of Abi1 in CHD1 cells, while both cell lines express comparable amounts of PI3K (I). There is slightly stronger Akt phosphorylation in CHD1 compared to HDC9 cells. Application of 50 nM Wortmannin (WO) almost completely repressed the pAkt and Abi1 signals. Immunofluorescence microscopy shows strong cytoplasmatic and nuclear Abi1 staining in CHD-1 cells, but only a faint cytoplasmatic signal in HDC-9 cells (II). <b>C,</b> KRAS/BRAF mutation testing reveals an activating KRAS G13D mutation in CHD-1 (left lane), while HDC-9 cells are KRAS wild-type (central lane). Transfection of HDC-9 cells with a KRAS G12D-construct leads to appearance of a band indicating a KRAS G12D-mutation (right lane). Both cell lines are BRAF wild-type. <b>D,</b> Immunoblotting of HDC-9 after transfection, TNFalpha and Wortmannin treatment shows an increase in pErk1/2 and pAkt and overexpression of Abi1 upon transfection with constitutively active KRAS G12D (2^nd lane) compared to both control (3^rd lane) and to transfection with wild-type KRAS (1^st lane). Stimulation with TNFalpha also enhances phosphorylation of signaling proteins and leads to upregulation of Abi1 (4^th lane). The overexpression of Abi1 could be reversed by application of 50 nM Wortmannin (5^th lane).</p

Comparison between results of the Idylla^™ KRAS Mutation Assay and of routine reference methods (codon level).

<p>Comparison between results of the Idylla^™ KRAS Mutation Assay and of routine reference methods (codon level).</p