Guanylate-Binding Proteins Promote Host Defense Against Leishmania major by Balancing iNOS/Arg-1 in Myeloid Cells

Abstract

Cutaneous leishmaniasis (CL) is a debilitating neglected tropical disease characterized by lesions that can range from self-healing to permanent disfigurations. 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, IFN-γ 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 (BMDMs) 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.

Keywords: Guanylate binding proteins; Leishmania; Leishmaniasis; Macrophages.

Figure 1:. GBP expression is a hallmark of CL.

Mice were infected intradermally with 2 × 10⁶ L. major parasites and at 4 weeks post infection (wpi) RNA was isolated from ear tissue and prepped for bulk RNA-Seq. (A) An MDS plot depicting up and down regulated transcripts during L. major infection. Red transcripts are upregulated with infection and blue transcripts are downregulated with infection. (B) Real-time PCR (RT-PCR) was conducted on RNA isolated from ear lesions from naïve mice or mice infected with 100,000 L. major parasites. Expression of GBPs during L. major infection was compared against the expression of each respective GBP in naïve mice to identify the fold change. (C) RT-PCR was performed on RNA isolated from ear lesions from mice infected with either 100,000 L. major, L. amazonensis, or L. mexicana at 6 wpi. Data in (B-C) is pooled from two independent experiments with 5 mice per group and results are shown as mean ± SEM. Significance was determined using a t-test compared to the naïve samples where *p<0.05 **p<0.01, ***p<0.001. Significance is shown in comparison to naïve samples.

Figure 2:. Macrophage GBPs control L. major parasites independent of localization to the parasite membrane.

BMDMs from control mice or GBP^Chr3 KO mice were infected and cultured in media or different supplemented media conditions before cells were prepped for cytospin analysis or immunofluorescence microscopy. (A) Representative images of control or GBP deficient macrophages infected with L. major and treated with IFN-γ for 72 hours. (B) Quantification of (A) showing parasites per macrophages and percent of infection after infection alone, infection and treatment with IFN-γ, or infection and treatment with LPS + IFN-γ after 72 hpi. (C-G) After 48 hpi, cells were fixed and stained with an antibody against GBP2 for immunofluorescence microscopy. Representative images of macrophages (C) cultured in media, (D) infected with L. major and only stained with a secondary antibody, (E) infected with L. major and stained with an antibody against GBP2, (F) IFN-γ-treated macrophages, or (G) IFN-γ-treated macrophages infected with L. major. (H) Quantification of the percentage of puncta containing cells as a percentage of total cells in (C-G) and (I) quantification of bystander versus infected cells containing puncta in (G). Data are representative of 2 or 3 independent experiments with at least 3 mice per group. Significance was determined using a two-way ANOVA paired with a Tukey’s multiple comparison test where *p<0.05 **p<0.01, ***p<0.001.

Figure 3:. GBPs participate in control of parasites and disease severity during L. major infection.

C57BL/6 control or GBP^Chr3 KO mice were infected intradermally with 2 × 10⁶ L. major parasites. (A) Diagram showing infection of control or GBP^Chr3 KO mice and representative lesions from each mouse strain at 2 wpi. (B) Lesions were monitored weekly by measuring ear thickness and lesion volume with electronic calipers. (C) The local ear and systemic dLN parasite burdens were quantified using a limiting dilution assay (LDA). Data is pooled from 3 experiments with 5 mice per group per experiment. Significance was determined using a t-test where *p<0.05 **p<0.01, and ***p<0.001.

Figure 4:. GBP expression is associated with dermal resident macrophage and monocyte-derived macrophage populations during L. major infection.

Mice were infected with L. major and at 4 wpi, scRNA-seq was performed on naïve ear skin and CL lesions. (A) UMAP plots depict 35 unique cell populations were identified in the ear dermis during L. major infection. (B) Feature plots of expression distribution for Gbp2, Gbp3, Gbp5, Gbp7 and Gbp9 in naïve animals and L. major infected mice. Expression levels for each gene are color-coded and overlaid onto UMAP plot. Cells with the highest expression level are colored dark purple. (C) Differential expression of selected GBP transcripts in 35 different cell types.

Figure 5:. GBP deficiency results in reduced iNOS⁺ myeloid cells and increased Arg-1⁺ myeloid cells.

Mice were infected with L. major parasites and at 2 wpi flow cytometric analysis was performed on ear tissue from control or GBP^Chr3 KO mice. (A) Representative flow plots of iNOS⁺ macrophages are shown. Gated on live, single, CD45⁺, CD11b⁺, CD64⁺, Ly6G⁻ cells. (B) Quantification of (A) shows percentage and number of iNOS⁺ macrophages in the infected and contralateral ear of control or GBP^Chr3 KO mice. (C) Representative flow plots of iNOS⁺ iMonos are shown. Gated on live, single, CD45⁺, CD11b⁺, Ly6C⁺, Ly6G⁻ cells. (D) Quantification of (C) shows percentage and number of iNOS⁺ iMonos in the infected and contralateral ear of control or GBP^Chr3 KO mice. (E) Representative flow plots of Arg-1⁺ macrophages are shown. (F) Quantification of (E) showing percentage and number of Arg-1⁺ macs. (G) Representative flow plots of Arg-1⁺ iMonos are shown. (H) Quantification of (G) showing percentage and number of Arg-1⁺ iMonos. (I) expression ratio of iNOS⁺ macrophages to Arg-1⁺ macrophages. (J) Expression ratio of iNOS⁺ iMonos to Arg-1⁺ iMonos. Data are pooled from 3 experiments with 5–10 mice per group per experiment. Data are shown as mean. Significance was determined using a two-way ANOVA paired with a Tukey’s multiple comparison test where *p<0.05 **p<0.01, ***p<0.001.