RasGEF1b regula a dinâmica do citoesqueleto de actina em macrófagos e a resposta inflamatória durante a infecção experimental por Coronavírus murino (MHV-3)

Abstract

Macrophages are immune cells with remarkable phenotypic and functional plasticity, regulated by the dynamics of the actin cytoskeleton, which controls essential processes such as motility, immune surveillance, and interactions with pathogens. Viruses, including coronaviruses, exploit this cellular machinery to promote their dissemination. Actin remodeling is coordinated by Rho GTPases, whose activation depends on guanine nucleotide exchange factors (GEFs). Preliminary data from our group revealed that RasGEF1b, a GEF specific to Ras proteins and highly expressed in macrophages, plays a role in maintaining cytoskeletal integrity, as its deficiency induces morphological alterations and cytoskeletal instability. In this study, we investigated the role of RasGEF1b in macrophage cytoskeletal organization and in the inflammatory response to experimental infection with murine hepatitis virus strain 3 (MHV-3). Using the RAW264.7 cell line with stable RasGEF1b knockdown via shRNA (RasGEF1b KD), we observed by confocal fluorescence microscopy that these macrophages exhibited cell retraction and reduced membrane ruffle formation, without impairments in macropinocytosis or phagocytosis. Additionally, RasGEF1b KD macrophages displayed reduced CCL2 expression under basal conditions and following LPS stimulation, which impaired their basal migratory capacity. Upon in vitro infection with MHV-3, RasGEF1b KD cells showed decreased formation of membrane protrusions and syncytia, structures associated with viral spread and immune evasion, resulting in reduced susceptibility to viral replication. In vivo, RasGEF1b knockout (KO) mice lacking the CMV-Cre transgene were generated. Following experimental MHV-3 infection, KO and wild-type (WT) mice exhibited similar total leukocyte counts and relative proportions of circulating granulocytes and lymphocytes. However, KO mice presented a lower neutrophil-to-lymphocyte ratio (NLR), significantly reduced pulmonary viral titers, and attenuated lung tissue damage, as evidenced by histopathological analysis. Bronchoalveolar lavage fluid (BALF) analysis revealed decreased leukocyte infiltration and a lower proportion of neutrophils in KO mice compared to WT. Despite reduced lung injury, KO mice exhibited elevated levels of proinflammatory cytokines, along with increased levels of the anti-inflammatory cytokine IL-10, suggesting that RasGEF1b deficiency modulates the inflammatory response in a manner that favors tissue protection. Collectively, these findings demonstrate that RasGEF1b plays a critical role in the structural organization of the actin cytoskeleton in macrophages and influences both inflammatory responses and susceptibility to coronavirus infection. The absence of RasGEF1b reduces viral replication and mitigates lung injury, underscoring its potential as a modulatory target of innate immunity in the context of viral infections.