  Citation: Alegre, F.; Martí-Rodrigo, A.; Polo, M.; Ortiz-Masiá, D.; Bañuls, C.; Pinti, M.; Álvarez, Á.; Apostolova, N.; Esplugues, J.V.; Blas-García, A. Macrophages Modulate Hepatic Injury Involving NLRP3 Inflammasome: The Example of Efavirenz. Biomedicines 2022, 10, 109. https://doi.org/10.3390/ biomedicines10010109 Academic Editor: Galina F. Sud’ina Received: 23 November 2021 Accepted: 30 December 2021 Published: 5 January 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). biomedicines Article Macrophages Modulate Hepatic Injury Involving NLRP3 Inflammasome: The Example of Efavirenz Fernando Alegre 1,2 , Alberto Martí-Rodrigo 1 , Miriam Polo 1,2 , Dolores Ortiz-Masiá 3,4 , Celia Bañuls 2 , Marcello Pinti 5 , Ángeles Álvarez 1,4, * , Nadezda Apostolova 1,2,4 , Juan V. Esplugues 1,2,4,† and Ana Blas-García 2,4,6,† 1 Departamento de Farmacología, Facultad de Medicina, Universidad de Valencia, 46010 Valencia, Spain; fernando.alegre@uv.es (F.A.); alberto.marti-rodrigo@uv.es (A.M.-R.); miriam.polo@uv.es (M.P.); nadezda.apostolova@uv.es (N.A.); juan.v.esplugues@uv.es (J.V.E.) 2 Servicio de Endocrinología, FISABIO-Hospital Universitario Dr. Peset, 46017 Valencia, Spain; celia.banuls@uv.es (C.B.); ana.blas@uv.es (A.B.-G.) 3 Departamento de Medicina, Facultad de Medicina, Universidad de Valencia, 46010 Valencia, Spain; m.dolores.ortiz@uv.es 4 Centro de Investigación Biomédica en Red Enfermedades Hepáticas y Digestivas (CIBERehd), 46010 Valencia, Spain 5 Department of Life Sciences, University of Modena and Reggio Emilia, 41121 Modena, Italy; marcello.pinti@unimore.it 6 Departamento de Fisiología, Universidad de Valencia, 46010 Valencia, Spain * Correspondence: angeles.alvarez@uv.es; Tel.: +34-963864898 † These authors contributed equally to the work. Abstract: Drug-induced liver injury (DILI) constitutes a clinical challenge due to the incomplete characterization of the mechanisms involved and potential risk factors. Efavirenz, an anti-HIV drug, induces deleterious actions in hepatocytes that could underlie induction of the NLRP3 inflammasome, an important regulator of inflammatory responses during liver injury. We assessed the potential of efavirenz to modulate the inflammatory and fibrogenic responses of major liver cell types involved in DILI. The effects of efavirenz were evaluated both in vitro and in vivo. Efavirenz triggered inflammation in hepatocytes, in a process that involved NF-κB and the NLRP3 inflammasome, and activated hepatic stellate cells (HSCs), thereby enhancing expression of inflammatory and fibrogenic markers. The NLRP3 inflammasome was not altered in efavirenz-treated macrophages, but these cells polarized towards the anti-inflammatory M2 phenotype and displayed upregulated anti-inflammatory mediators. Conversely, no evidence of damage was observed in efavirenz-treated animals, except when macrophages were depleted, which resulted in the in vivo manifestation of the deleterious effects detected in hepatocytes and HSCs. Efavirenz elicits a cell-specific activation of the NLRP3 inflammasome in hepatocytes and HSCs, but macrophages appear to counteract efavirenz- induced liver injury. Our results highlight the dynamic nature of the interaction among liver cell populations and emphasize the potential of targeting macrophage polarization as a strategy to treat NLRP3 inflammasome-induced liver injury. Keywords: antiviral therapy; inflammation; fibrogenesis; DILI; macrophage polarization 1. Introduction Dysregulated inflammatory responses contribute to the pathogenesis of most acute and chronic liver diseases, including drug-induced liver injury (DILI), which constitutes a clinical challenge due to the difficulties to characterize the mechanisms involved and the risk factors responsible for its worsening [1]. Excessive inflammation is crucial in drug-induced hepatic damage and, when associated with hepatotoxic insults, it can lead to fibrosis [2]. Hepatic inflammation and fibrogenesis are mediated by the inflammatory path- way regulated by NF-κB and by inflammasomes, intracellular complexes that recognize Biomedicines 2022, 10, 109. https://doi.org/10.3390/biomedicines10010109 https://www.mdpi.com/journal/biomedicines