A Single Intravenous Dose of Indole-producing Attenuated Brucella in NOD Mice: Assessment of Type 1 Diabetes Onset and Islet Immune Remodeling

Summary

This protocol describes intravenous administration of an indole-producing attenuated Brucella melitensis strain to NOD/ShiLtJ mice, followed by longitudinal diabetes monitoring, pancreatic histology, and optional high-plex spatial proteomics and single-cell RNA sequencing to assess islet immune reprogramming.

Abstract

Type 1 diabetes (T1D) results from autoimmune destruction of pancreatic β cells, and current therapies fail to restore durable immune tolerance. Metabolically engineered bacteria producing immunomodulatory metabolites represent a promising single-dose therapeutic strategy. Here, this paper presents a comprehensive protocol in which a single intravenous administration of an indole-producing attenuated Brucella melitensis strain BmΔvjbR::tnaA is used to assess effects on the onset of autoimmune diabetes in prediabetic female NOD/ShiLtJ mice.

The protocol begins with preparing BmΔvjbR::tnaA from frozen glycerol stocks, growing it in selective media to mid-log phase, and calculating colony-forming units based on optical density. Female NOD mice, <5-week-old are restrained without anesthesia and injected intravenously via lateral tail vein with a defined bacterial dose in phosphate-buffered saline while maintained on heating pads. Blood glucose is monitored 2x weekly after a 2 h fast for 70–105 days, with diabetes defined as glucose ≥250 mg/dL on two consecutive readings. At experimental endpoints, harvested pancreas are fixed in neutral-buffered formalin for 72 h, paraffin-embedded, and sectioned for hematoxylin and eosin staining, insulin immunohistochemistry, and total islet area quantification. Formalin-fixed paraffin-embedded sections are submitted for spatial proteomics with a 25-marker antibody panel to visualize regulatory and effector immune populations. Finally, fresh pancreatic tissue undergoes single-cell isolation, 3′ library preparation, sequencing, and Seurat-based bioinformatics analysis to profile transcriptional changes across immune and stromal populations.

This integrated protocol enables reproducible single-dose bacterial therapy delivery and multi-modal characterization of islet immune remodeling, providing a framework for testing metabolite-engineered microbes in autoimmune models and dissecting tissue-specific immune mechanisms at single-cell resolution.

Introduction

Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by immune-mediated destruction of insulin-producing pancreatic β cells, leading to lifelong insulin dependence and increased risk of vascular complications¹. The NOD/ShiLtJ mouse recapitulates key features of human autoimmune diabetes, including spontaneous insulitis and progression to overt diabetes, and is widely used as a preclinical model for mechanistic and therapeutic studies^1,2,3. Current T1D therapies—including immunosuppressive drugs, monoclonal antibodies, and adop....

Protocol

All animal experiments were conducted in strict accordance with ethical guidelines and approved by the Institutional Animal Care and Use Committee (IACUC) of Texas A&M University (Protocol Number: 2021-0123).

1. Preparation and characterization of BmΔvjbR::tnaA

1. Bacterial strain background and storage
   ​NOTE: BmΔvjbR::tnaA is derived from Brucella melitensis 16M with deletion of the vjbR virulence regulator and insertion of the Escherichia coli tnaA gene for constitutive indole production, as previously described^14,

Results

While the broader protocol described in this manuscript has since been optimized into a single-dose preventive regimen, these pilot outcomes are presented here solely to illustrate the expected histological and glycemic readouts achievable with this workflow.

The overall disease context and contrast between healthy and type 1 diabetic pancreas are illustrated schematically in Figure 1A. The autoimmune destruction of β cells and resulting loss of insulin production.......

Discussion

A critical limitation of the representative outcomes presented herein is that the data provided to illustrate the analytical endpoints (Figure 3) were derived from an early pilot cohort utilizing a two-dose schedule. Because this protocol has been refined to a single-dose preventative regimen, future larger-scale studies utilizing the optimized single-dose protocol described here are required to establish formal statistical efficacy.

This JoVE protocol describes a.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10% Neutral Buffered Formalin (NBF)	VWR	10790-714
10x Genomics Chromium Controller	TIGSS Molecular Genomics Workspace, Texas A&M University
70% Ethanol	VWR	89125-164
Adhesive microscope slides for FFPE	Leica Biosystems	3800084E
Akoya mouse FFPE antibody panel (~25 markers)	Akoya Biosciences	[Panel ID/custom panel]
Bioanalyzer or TapeStation with DNA HS kits	TIGSS Molecular Genomics Workspace, Texas A&M/ Agilent
BmΔvjbR::tnaA bacterial strain	Lab stock (Song/de Figueiredo labs)
Cell Ranger pipeline	10x Genomics	v7.1
Chloramphenicol	Research Products International	56-75-7
Chromium Next GEM Single Cell 3' kit	10x Genomics via TIGSS core
Collagenase from Clostridium histolyticum	Millipore Sigma	C7657-100MG
DAPI nuclear stain	Akoya Biosciences	[Service-provided]
FFPE embedding, H&E staining, insulin IHC, and slide scanning	VMBS Histology Laboratory, Texas A&M University	https://vetmed.tamu.edu/vmbs-histology-lab/
Glucose test strips	EKF Diagnostics / Fisher Scientific	22-022-649
Glucose test strips (alternate)	Ascensia Diabetes Care	CONTOUR NEXT test strips
Glycerol (bulk, 500 mL)	Millipore Sigma	G7893-500ML
Glycerol (sterile, 100 mL)	Sigma Aldrich (G Biosciences)	G5516-100ml
GraphPad Prism (or R survival package)	GraphPad Software / R	v10.2
Handheld glucometer	Ascensia Diabetes Care	CONTOUR NEXT EZ
Illumina NextSeq 2000 sequencer	TIGSS Molecular Genomics Workspace, Texas A&M University
Insulin syringes,29–30 G (U-100)	VWR	10002-702
Lancets for diabetes testing	Becton, Dickinson and Company (BD)	366594
Liberase TL Research Grade	Millipore Sigma	5401020001
Low-temperature heating pads	Conduct Science	RWD-69025
Miltenyi nuclei/cell isolation kit	TIGSS Molecular Genomics Workspace, Texas A&M/ Miltenyi Biotec
Mouse heating pad (alternate)	Kent Scientific	RightTemp
Mouse restrainers	Braintree Scientific	TV-RED 150-STD
NOD/ShiLtJ mice (female, 5 weeks)	The Jackson Laboratory	Strain #001976
P2 XLEAP 100-cycle flow cell kit	Illumina via TIGSS core
PhenoCycler data acquisition and QC service	Akoya Biosciences
PhenoCycler reagents and imaging workflow	Akoya Biosciences	[Service-provided]
PhenoCycler-Fusion system	Akoya Biosciences
Phosphate-buffered saline (PBS), 1x	Thermo Fisher	10010023
Protector RNase Inhibitor	Millipore Sigma	3335402001
Qubit Fluorometer and dsDNA HS Assay Kit	TIGSS Molecular Genomics Workspace, Texas A&M/ Thermo Fisher
QuPath	Open source	v0.4.3
R software	R Project	v4.0+
Seurat (R package)	Satija Lab / CRAN	v4.3
StarDist	Open source	v0.8.3
Tryptone Soy Agar (TSA)	VWR	CA90002-706