Guanylate-binding proteins balance iNOS/Arg-1 in myeloid cells during L. major infection and promote host defense to infection.

Cutaneous leishmaniasis (CL) is a debilitating neglected tropical disease characterized by lesions that can range from self-healing to permanent disfiguration. A predominant Th1 response, which stimulates IFN-γ production, is crucial for parasite control during self-healing CL. While IFN-γ primarily activates macrophages to produce nitric oxide via inducible nitric oxide synthase (iNOS), leading to parasite control, it also activates other downstream pathways involved in cell autonomous immunity. One such pathway is the activation of guanylate-binding proteins (Gbps), a class of interferon-inducible GTPases. However, the role of Gbps during CL has been minimally explored. Utilizing RNA-Seq, we found that Leishmania major infection leads to the upregulation of several Gbps in C57BL/6 mice. In vitro studies using GbpChr3 knockout (KO) and C57BL/6 control mice reveal that bone marrow-derived macrophages from KO mice exhibit higher parasite burdens following IFN-γ treatment, independent of Gbp localization to the parasite. Single-cell RNA-Seq identifies macrophages as the primary expressers of Gbps during L. major infection in vivo. In vivo, GbpChr3 KO mice display increased disease severity and parasite load. GbpChr3 KO macrophages and monocytes show elevated ARG-1 and reduced iNOS expression, indicating a shift toward a parasite-permissive environment that supports parasite growth. These findings highlight a critical role for Gbps in immune-mediated control of CL.IMPORTANCELeishmania parasites cause cutaneous lesions that are often resistant to drug treatment, and no vaccine is currently available, highlighting the need to better understand host mechanisms that control infection. In this manuscript, we explore the role of guanylate-binding proteins (Gbps) in host macrophages during Leishmania major infection. We demonstrate that Gbps are critical for host defense both in vitro and in vivo. Notably, this protection is independent of Gbp localization to the parasite, revealing a novel aspect of Gbp biology. Instead, we find that differences in parasite burden and disease severity in Gbp-deficient mice are linked to altered activation of tissue macrophages and monocytes. Our findings suggest that Gbps coordinate inducible nitric oxide synthase expression in macrophages, the primary cells that house and control Leishmania parasites, and play a unique immunoregulatory role during infection.