Inducible nitric oxide synthase (iNOS) regulates skin eschar lesions, bacterial persistence, and inflammatory resolution in mouse models of scrub typhus

Description

Orientia tsutsugamushi (Ot) is an obligately intracellular bacterium that causes scrub typhus, a potentially severe infectious disease characterized by systemic inflammation and multiorgan dysfunction. We recently reported an essential role for IFN-γ signaling in host defense against Ot infection; however, its underlying mechanisms remain incompletely understood. Inducible nitric oxide synthase (iNOS, encoded by Nos2) is a key antimicrobial effector induced downstream of IFN-γ signaling. Here, we used transgenic mouse models to further investigate the biological functions of iNOS. We first revealed the requirement of iNOS for the restriction of Ot growth in cultured bone marrow-derived macrophages. Using an intradermal mouse model, we found that while tissues of Nos2⁻/⁻ and wild-type mice exhibited comparable bacterial burdens during acute infection phases, Nos2⁻/⁻ mice developed skin eschar-like lesions similar to those observed in Ifngr1⁻/⁻ mice, indicating a critical role for the IFN-γ/iNOS axis in regulating skin pathology in scrub typhus. Notably, Nos2⁻/⁻ mice displayed impaired bacterial clearance during the recovery phase, with persistent bacterial burdens in multiple organs accompanied by sustained immune activation and elevated inflammatory responses. Histopathological and biochemical analyses further revealed increased tissue damage and dysregulated physiological homeostasis in the Nos2-/- mice during recovery. Mechanistically, iNOS deficiency resulted in heightened myeloid cell activation and prolonged expression of proinflammatory mediators, suggesting a dual contribution of iNOS in both antimicrobial defense and inflammation resolution. Collectively, these findings provide new insight into IFN-γ-mediated defense mechanisms and imply the distinct roles of iNOS during different stages of scrub typhus. Graphpad Prism is used to perform data statistical analysis and graph generation. The raw data presented as figures in the PPT are accessible. All HE and flow cytometry raw data are uploaded separately.

Steps to reproduce

For animal infection, mice were first anesthetized in a chamber connected to a VetFlo isoflurane vaporizer. Next, mice were carefully removed from the chamber, and the infection site (right flank) was shaved using an electric trimmer. Mice were inoculated with Ot Karp strain (2×10e3 FFU, 20 µL volume) in the dermis of the flank by using a 0.3 mL insulin syringe with 31-G needles. Bacterial burdens were quantified from harvested mouse tissues and cultured cells. DNA was then extracted using the DNeasy Blood and Tissue Kit (Qiagen) and subjected to qPCR analysis as previously described. The 47-kDa gene was amplified using the primer pair OtsuF630 (5′-AACTGATTTTATTCAAACTAATGCTGCT-3′) and OtsuR747 (5′-TATGCCTGAGTAAGATACGTGAATGGAATT-3′) (IDT). Detection was performed with the probe OtsuPr665 (5′-6FAM-TGGGTAGCTTTGGTGGACCGATGTTTAATCT-TAMRA-3′) (IDT) using SsoAdvanced Universal Probes Supermix (Bio-Rad). Absolute quantification was determined using a 10-fold serial dilution of an Ot Karp 47-kDa plasmid standard. Total RNA was extracted from mouse lung and brain tissues using the RNeasy Mini Kit (Qiagen). cDNA was synthesized from 1 μg of total RNA using the iScript Reverse Transcription Kit (Bio-Rad). qRT-PCR was performed in a total reaction volume of 10 μL containing 5 μL of iTaq SYBR Green Supermix (Bio-Rad), 1 μL of forward and reverse primer mix (final concentration 0.5 μM each), and 4 μL of diluted cDNA. Amplification was carried out on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad) with the following cycling conditions: initial denaturation at 95°C for 30s, followed by 40 cycles of 95°C for 15s and 60°C for 60s. Relative gene expression levels were calculated using the 2−ΔΔCt method and normalized to Gapdh. Animal blood chemistry analysis was performed by using the VetScan Chemistry Analyzer (Zoetis, Parsippany-Troy Hills, NJ). Spleen single-cell suspensions were prepared directly by pressing spleen tissues through 70-μm cell strainers using a sterile 3-cc syringe. Equivalent portions of lung tissues were harvested from mice, minced, and digested with 0.05% collagenase type IV (Thermo Fisher Scientific) in RPMI-1640 medium for 30 min at 37°C. Minced tissues were loaded into Medicons and homogenized using a BD Mediamachine System (BD Biosciences). Lung single-cell suspensions were made by passing lung homogenates through 70-μm cell strainers. Red blood cells were removed by using Red Cell Lysis Buffer (Sigma) for 5 min at room temperature. For surface marker analysis, leukocytes were stained with the Fixable Viability Dye (eFluor 506, Thermo Fisher Scientific) for live/dead cell discrimination, blocked with FcγR blocker, and incubated with fluorochrome-labeled antibodies for 30 min at 4°C.

Institutions

• The University of Texas Medical Branch at Galveston

  Texas, Galveston

Categories

Funders

• National Institute of Allergy and Infectious Diseases

  National Institutes of Health

  Maryland, Bethesda

  Grant ID: AI132674, AI179997, AI184781

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