Ethics Statement
All animal studies were directed in accordance to protocols approved by the Center for Infectious Disease Research (now Seattle Children’s Research Institute; CIDR protocol DS-05 UW), and Washington National Primate Research Center, Seattle, WA (protocols 4314–01, 4213–02 and 4213–03) under the Institutional Animal Care and Use Committees (IACUCs). All rhesus macaques involved in this study were managed according to the laws, regulations, and guidelines set forth by the United States Department of Agriculture, Institute for Laboratory Animal Research, Public Health Service, National Research Council, Centers for Disease Control, the Weatherall Report titled “The use of nonhuman primates in research”, and the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International. Nutritional plans utilized by WaNPRC consisted of standard monkey chow supplemented with a variety of fruits, vegetables, and other edible objects as part of the environmental enrichment program established by the Behavioral Management Unit. Enrichment was distributed and overseen by veterinary staff with animals having access to more than one category of enrichment. Uninfected macaques exhibiting incompatible behaviors were managed by the Behavioral Management staff and managed accordingly. SIV + macaques were kept in individual, adjoining cages allowing for social interactions with primate health observed daily by trained staff. All efforts were made to minimize suffering using minimally invasive procedures, anesthetics, and analgesics when deemed appropriate by veterinary staff. Animals were painlessly euthanized by sedation with ketamine hydrochloride injection followed by intravenous barbiturate overdose following the recommendations of the panel of euthanasia of the American Veterinary Medical Association. These macaques have been described previously [32].
Liver Tissue Collection
Liver tissue was collected at necropsy from uninfected (N = 4), SIV+ (N = 6) and SIV + cART (N = 6) adult Indian rhesus macaques (Macaca mulatta). Control samples from uninfected macaques were acquired from the Tissue Donor Program at WaNPRC. SIV + macaques were infected intrarectally with SIVmac239x [32]. Macaques receiving cART were administered subcutaneous tenofovir (20 mg/kg body weight) and emtricitabine (30 mg/kg) and oral raltegravir (50 mg twice daily) starting 120 days post-infection and continuing for 35–36 weeks prior to euthanasia [32]. Tissue was formalin-fixed, paraffin-embedded for microscopy or flash-frozen in liquid nitrogen and then stored at -80 °C for nucleic acid extraction.
Immunofluorescence Staining
Liver tissue was obtained at necropsy (14–55 weeks post infection), fixed in 10% formalin, and paraffin-embedded. Slides were prepared from 5µ m tissue sections and dewaxed with xylene and rehydrated with a gradient of ethanol baths. Antigen retrieval was performed with a 1% citrate buffer (Antigen Unmasking Solution, Vector Laboratories, Burlingame, CA) in a decloaking chamber at 90 °C for 30 minutes with a 10-minute cooldown. Slides were washed in a series of TBST (0.025% Triton X-100 in 1X TBS) followed by a 2-hour block step (0.1% BSA, 1% goat serum in TBST). Tissues were stained overnight at 4 °C for mouse anti-human myeloperoxidase (MPO, polyclonal, 1:2000, Dako) and rabbit anti-human CD68 (clone KP1, 1:250, Santa Cruz Biotechnology, Dallas, TX). Slides were then washed in another series of TBST washes and incubated with secondary antibody for one hour in the dark using AlexaFluor 488 goat anti-mouse (1:500, Life Technologies, Carlsbad, CA) to detect MPO + neutrophils and AlexaFluor 594 goat anti-rabbit (1:500, Life Technologies) to detect CD68 + macrophages. After incubation, slides were washed in a series of TBST washes and mounted with Vectashield Hard set DAPI (Vector Technologies) and allowed to set. For each liver section, eight random fields were imaged at 200x magnification. Cells were counted using ImageJ.
Liver Tissue Disruption By Pulverization
Flash-frozen liver tissue was pulverized into a fine powder by ball milling with stainless balls under cryogenic conditions with liquid nitrogen (Retsch Planetary Ball Mill, Retsch Laboratory Equipment, Haan, Germany). Each sample was subjected to three cycles at 300 rpm for two minutes each. Following pulverization, the liver powder was collected and stored at -80 °C until DNA extraction.
Tissue DNA Extraction from Liver Powder
Liver powder (10–30 mg) was placed into a sterile, pre-chilled microcentrifuge tube. Genomic DNA was extracted using the NucleoSpin Tissue DNA extraction kit (Takara, Mountain View, CA) per the manufacturer’s instructions, where samples were pre-lysed and allowed to incubate at 56°C for at least 1–3 hours vortexing occasionally. Samples were then lysed with provided buffer, vortexed vigorously, and incubated at 70°C for 10 minutes. Ethanol was added and samples were centrifuged in NucleoSpin Tissue Columns into a collection tube at 11,000 x g for 1 minute. After a series of washes, samples were eluted with elution buffer and collected. Following concentration determination with a NanoDrop 2000 Spectrophotometer (Thermo Scientific, Waltham, MA), isolated genomic DNA was stored at -80 °C until use.
16s rRNA Gene Sequencing and Microbiome Analysis
Genomic DNA extracted from the liver (20 µL) was used for 16 s rRNA gene sequencing through Illumina according to the EMP method. In brief, a 460-bp amplicon was generated targeting the V3-V4 region of the 16 s rRNA gene. PCR amplicons were cleaned with 0.8x AMPure XP beads (Beckman Coulter, Brea, CA) before the addition of Nextera XT dual index adaptors (Illumina Inc., San Diego, CA). Indexed amplicons were cleaned using 1.1 × AMPure XP beads (Beckman Coulter), quantified using a Qubit DNA high-sensitivity assay kit (Life Technologies), and multiplexed using an equal molar ratio of DNA for each sample. 16S rRNA gene libraries were loaded on a 300-cycle MiSeq kit and sequenced using Nextera sequencing read and index primers (all from Illumina Inc.). Paired-end demultiplexed FASTQ files from the Illumina base space were imported into the QIIME2 pipeline (QIIME 2 Core 2019.10) to create a demultiplexed QIIME2 object. These objects were matched to identified amplicon sequence variants (ASVs) using the dada2 algorithm which worked to detect and correct Illumina amplicon sequence data and denoise by trimming to 145 bases to remove low-quality regions. A rooted phylogenetic tree was constructed using the Mafft multiple sequencing alignment program and taxonomy was assigned using the Greengenes database specific to the V3-V4 region. After taxonomy was determined, results were exported from the pipeline for downstream analysis in R using the phyloseq package.
Quantification of Mycobacterial DNA in the Liver by qPCR
All liver DNA samples were diluted in nuclease-free water. Each sample (5 µL) was prepared in duplicate in a 20 µL volume reaction with the PowerUp SYBR Green Master Mix kit (Applied Biosystems, Waltham, MA) and Mycobacterium-specific primers (MycoARB210: TTT GCG GTG TGG GAT GGGC and MycoARB585: CGA ACA ACG CGA CAA ACCA). A ‘No Template’ Negative Control was included to control for contamination and non-specific amplification. A standard curve was generated by serially diluting pure M. bovis (BCG) DNA 10-fold, ranging from 10-0.001 ng/µL (R2 > 0.95). PCR reactions ran one cycle at 50 °C for 2 minutes then increasing to 95 °C for 2 minutes followed by 45 cycles of 94 °C for 15 seconds, annealing at 61 °C for 30 seconds, and extending at 72 °C for 30 seconds with a final extension step at 72 °C for 7 minutes. Following the qPCR cycles, PCR reactions were subjected to a melt curve analysis to examine products formed. The concentration of Mycobacterium per sample was determined through a non-linear regression on the standard curve and converted to copy number based on BCG molecular weight (5.63 × 1012 mg/mole). The weight was then converted to 4.277 × 107 molecules/mole and the standard curve was plotted based on molecules where copy number was equal to 4.277 × 107 molecules/mole * log (CT) where the standard curve equation was extrapolated (y = -0.032ln(x) + 1.8377). Copy number of the liver Mycobacterium DNA was then calculated from the standard curve equation (copy number = e ((log (Ct) – 1.8377)/-0.032)). Duplicates were averaged for each animal. qPCR to detect a conserved region of the 16S rRNA gene was performed as reported previously [32].
Identification of Mycobacterium Species by Multi-Gene Sequencing
Genomic DNA (extracted as described above) was diluted in nuclease-free water and amplified by nested PCR per the conditions outlined in Additional File 1. For each first-round PCR reaction, 500 ng (5 µL) of gDNA was added into a 50 µL reaction and amplified using the Platinum Taq DNA Polymerase reaction kit (Invitrogen, Carlsbad, CA). For nested PCR reactions, 1 µL of the first-round PCR product was added to a 50 µL reaction containing the nested primers and Platinum Taq DNA Polymerase. Each round of PCR contained a positive control of BCG DNA and a negative no template control. Following nested PCR, each reaction was examined on a 1% agarose gel. Each PCR amplicon showing the correct size was cleaned up using a Nucleospin PCR Clean-up Kit (Takara) and eluted into 30 µL of EB buffer. Purified PCR amplicons (20 ng) were sent for Sanger sequencing using both forward and reverse nested primers in separate reactions. Following sequencing, DNA sequence quality was examined in 4Peaks software and low-quality reads from the 5’ and 3’ ends removed. Consensus sequences generated through the alignment of forward and reverse reads were analyzed using BLAST analysis.
Culture and Stimulation of Human Hepatocytes with Mycobacteria and Mycobacterial Antigens
Human HepaCure Hepatocytes on Matrigel overlay (350,000 hepatocytes/well) were acquired from Yecuris (Tualatin, OR) in 24-well dishes. HepaCure human hepatocytes are produced by the immunization of humanized FRG®KO mice with cadaver-derived human hepatocytes. Upon receipt, the media was immediately replenished with 500 µL InVitro GRO Hi Medium (BioIVT, Westbury, NY) supplemented with Torpedo Antibiotic Mix (BioIVT). Cultures were incubated at 37 °C, 5% CO2 overnight. To determine the hepatocyte response to mycobacterial pathogen associated molecular patterns (PAMPs), M. smegmatis purified lipoarabinomannan (PILAM, 0.1 and 10 ug/mL, BEI Resources, Manassas, VA), or M. tuberculosis, Strain H37Rv, purified lipoarabinomannan (ManLAM, 0.1 and 10 ug/mL, BEI Resources), were added to hepatocytes. Each stimulation condition was conducted in triplicate. Plates were incubated at 37 °C, 5% CO2 for 24 hours. For live mycobacteria stimulations, M. smegmatis bacteria (strain MC2155) were grown to exponential phase, washed with PBS and resuspended in InvitroGRO Hi Medium without antibiotics at 350,000 bacteria/µL. Hepatocytes were stimulated with M. smegmatis (MOI 10) in duplicate for 24 hours at 37 °C, 5% CO2. For all stimulations, conditioned media was collected and stored at -80 °C. The hepatocyte monolayer was then washed with 500 µL pre-warmed PBS and then lysed in 300 µL RA1 buffer containing beta-mercaptoethanol. RNA was isolated from the cell lysate following protocols from the NucleoSpin RNA isolation kit (Macherey-Nagel, Bethlehem, PA).
Transcriptomic Analysis of HepaCure Hepatocytes by Nanostring
RNA was diluted to 20 ng/µL in nuclease-free water and used for transcriptomic analysis using a Nanostring Inflammation Panel (Human v2) (Nanostring, Seattle, WA). Probe set-target RNA hybridization reactions were performed according to the manufacturer’s protocol using 100 ng (5 µL) of total RNA. Purified probe set-targets were processed and immobilized on nCounter Cartridges using a nCounter MAX prep station. Transcripts of interest were quantified on the Digital Analyzer for each sample. For data analysis, nCounter RCC files were imported in nSolver Analysis Software 4.0 and checked for quality control. Determination of differentially expressed genes, pathways analysis, and cell profiling was conducted using the Nanostring Advanced Analysis software per the manufacturer’s instructions. For each stimulation condition, differentially expressed genes were determined by comparing the normalized count data between stimulated hepatocytes and unstimulated control hepatocytes. Heatmaps were generated in Prism version 5.0f software (GraphPad Software, Inc., San Diego, CA), showing fold change of each gene in the panel. Volcano plots were assessed using the python matplotlib package for significant genes using a threshold of 1.5-fold change (log2(1.5) = 0.585) and 0.05 adjusted p-value.
Statistics
Statistical analyses were performed using Prism version 5.0f software (GraphPad Software, Inc.). A nonparametric Mann-Whitney U test was used to compare the SIV + and SIV + cART groups to the uninfected controls. Linear regression and Spearman correlation analyses were performed. Analysis of gene expression panels was completed using nSolver (Nanostring, version 4.0.62).