Neutrophils insert elastase into hepatocytes to regulate calcium signaling in alcohol-associated hepatitis
Noriyoshi Ogino, … , Barbara E. Ehrlich, Michael H. Nathanson
Noriyoshi Ogino, … , Barbara E. Ehrlich, Michael H. Nathanson
Published June 25, 2024
Citation Information: J Clin Invest. 2024;134(16):e171691. https://doi.org/10.1172/JCI171691.
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Research Article Hepatology

Neutrophils insert elastase into hepatocytes to regulate calcium signaling in alcohol-associated hepatitis

Abstract

Neutrophil infiltration occurs in a variety of liver diseases, but it is unclear how neutrophils and hepatocytes interact. Neutrophils generally use granule proteases to digest phagocytosed bacteria and foreign substances or neutralize them in neutrophil extracellular traps. In certain pathological states, granule proteases play a destructive role against the host as well. More recently, nondestructive actions of neutrophil granule proteins have been reported, such as modulation of tissue remodeling and metabolism. Here, we report a completely different mechanism by which neutrophils act nondestructively, by inserting granules directly into hepatocytes. Specifically, elastase-containing granules were transferred to hepatocytes where elastase selectively degraded intracellular calcium channels to reduce cell proliferation without cytotoxicity. In response, hepatocytes increased expression of Serpin E2 and A3, which inhibited elastase activity. Elastase insertion was seen in patient specimens of alcohol-associated hepatitis, and the relationship between elastase-mediated ITPR2 degradation and reduced cell proliferation was confirmed in mouse models. Moreover, neutrophils from patients with alcohol-associated hepatitis were more prone to degranulation and more potent in reducing calcium channel expression than neutrophils from healthy individuals. This nondestructive and reversible action on hepatocytes defines a previously unrecognized role for neutrophils in the transient regulation of epithelial calcium signaling mechanisms.

Authors

Noriyoshi Ogino, M. Fatima Leite, Mateus T. Guerra, Emma Kruglov, Hiromitsu Asashima, David A. Hafler, Takeshi Ito, João P. Pereira, Brandon J. Peiffer, Zhaoli Sun, Barbara E. Ehrlich, Michael H. Nathanson

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Figure 1

Neutrophils decrease Ca2+-related proteins in hepatocytes without causing cell death.

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Neutrophils decrease Ca2+-related proteins in hepatocytes without causin...
(A–C) Representative images of HepG2 cells and neutrophils after 20 hours of culture, using double staining (calcein-AM for alive [green], ethidium homodimer-1 for dead [red]). HepG2 cells only (A), coculture (B), neutrophils only (C). (D and E) Graphs comparing time series of cell viability with and without coculture. (D) Viability of HepG2 cells does not decrease over time regardless of coculture. (E) Viability of neutrophils decreases over time unless they are cocultured. Differences between alone and coculture are represented by daggers, and changes over time are indicated by asterisks; 5 fields per coverslips were measured. (F and G) Representative images double labeled with EdU (green) and Hoechst 33342 (blue) of HepG2 cells alone (F) and cocultured with neutrophils (G) after 12 hours. (H) HepG2 cell proliferation (EdU-positive cells) is decreased by coculture with neutrophils; 5–6 fields were measured per coverslip. (I) Ca2+ signals in HepG2 cells are altered by neutrophils. Representative tracings of Fluo-4 fluorescence intensity of HepG2 cells alone or cocultured with neutrophils for 20 hours. Cells were stimulated with 20 μM ATP. (J) Ca2+ signaling in HepG2 cells as measured by the AUC upon ATP stimulation is diminished by neutrophils. Nine coverslips of HepG2 cells (362 total cells) and 8 coverslips of HepG2 cells cocultured with neutrophils (279 total cells) were analyzed. (K) Representative immunoblots and (L) quantitation of the blots show that ITPR1, ITPR2, ITPR3, and SERCA2 are decreased in HepG2 cells, but calnexin and SEC61B are not, after coculture with neutrophils for 1 hour. ITPR1, ITPR2, and ITPR3 were blotted onto different membranes because of their close molecular weights, and their ratios to GAPDH were measured separately. (M) Representative immunoblots and (N) quantitation of the blots shows that ITPR2 and SERCA2 are decreased in primary human hepatocytes (HHC) that are cocultured for 1 hour. The graph represents neutrophils from 5 healthy volunteers. In A–N, all are from 3 independent experiments. Data are mean ± SD. NS, not significant. **P < 0.01; ****P < 0.0001; †††P < 0.001; ††††P < 0.0001 by unpaired, 2-tailed Student’s t test. Scale bars: 50 μm.