Transcriptome datasets of macrophages infected with different strains of Leptospira spp

The datasets reported herein provide information about microarray experiment of macrophage cell line J774A.1 infected with three different strains of Leptospira spp. Transcriptomic profiles were generated using Affymetrix® Mouse Gene 2.1 ST Array Strip. Data was normalized and statically process, p-value < 0.01, FDR < 0.05 and log2 fold change (± 2). The microarray raw data are available in Gene Expression Omnibus (GEO) under accession number GSE105141.

Data reported will help in understanding the pathogenicity and immune response to leptospirosis which is still poorly understood.
This data can reveal new insight on modulation and function of genes in immune cells following infection by Leptospira, with particular focus on differential host immune response to varying bacterial virulence.

Data
Total RNA was extracted from murine macrophage cell line J774A.1 infected with virulent, attenuated or saprophyte strains of Leptospira, as well as control non-infected cells, 6 h post in vitro infection. Affymetrix microarray was performed to obtain transcriptomic profiles of the infected and control groups. Raw data was deposited at NCBI GEO DataSets (GSE105141).

Bacterial culture
Samples of the virulent strain Leptospira interrogans sorovar Copenhageni (FIOCRUZ L1-130), attenuated strain L. interrogans sorovar Copenhageni M20, and saprophyte strain L.biflexa sorovar Patoc (FIOCRUZ -Patoc I) were utilized. Attenuation of sample M20 was performed with successive replications in culture medium. All strains were maintained in Fletcher semi-solid culture medium, and incubated at 30°C. To restore bacterial virulence (virulent strain), 1 mL of cultured bacteria was inoculated intraperitoneally in Golden Syrian hamsters (Mesocricetus auratus) and later recovered from kidneys, whereas the attenuated strain did not undergo intraperitoneal inoculation in hamsters. Virulent leptospires cultures are routinely maintained at the Faculdade de MedicinaVeterinária e Zootecnia, Universidade de São Paulo (USP), São Paulo, Brazil, as previously described [1]. The inoculum was quantified using a Petroff-Hausser chamber.

Infection of macrophages
After the formation of a confluent cell monolayer, cultured cells were washed tree times with sterile phosphate buffer solution (pH 7,2) for removal of antibiotics and non-adherent cells. L. interrogans and L. biflexa were harvested by centrifugation and the pellet was resuspended in RPMI-1640 media (Sigma), and 100:1 bacteria:cell of the three groups (virulent, attenuated and saprophyte) were added to macrophages and non-infected macrophages were kept as control. All treatments were performed in biological triplicates. Cells (infected and control) were incubated in fresh RPMI medium, without antibiotics, for 6 h at 37°C, 5% C02. Following this period, RNA extraction was immediately performed as described below.

RNA extraction and quantification
Total RNA was extracted from macrophages with a RNeasy Mini Kit (Qiagen, USA) according to manufacturer's instructions. RNA samples were immediately stored at −80°C. Quantification was performed using NanoDrop (ND-2000 spectrophotometer, Thermo Scientific, Wilmington, DE, USA) with OD260/280 ratios between 2,07 and 2,09 and concentration ranging of 1689 to 2818 ng/µl ( Table 1). Quality of samples was assessed using capillary electrophoresis (Bioanalyzer 2100 Agilent, Santa Clara, CA, USA) and all samples used for microarray analysis had a RIN of 10 ( Fig. 1a-b).

Microarray analysis
A WT PLUS Reagent kit (Affymetrix, Santa Clara, CA, USA) was used to prepare the RNA samples for whole transcriptome expression analysis using the Mouse Genome 2.1 ST Array Strip (Affymetrix), according to the manufacturer´s instructions. Briefly, Poly-A RNA controls (lys, phe, thr and dap), absent in eukaryotic cells, were added to the RNA samples (assay control) prior to generation of cDNA. After the amplification process, final cDNA was purified, quantified, fragmented and labeled for hybridization to strips (all 4 groups were represented in each strip, experiment was repeated three times). Strips remained for 20 h at 48°C in a hybridization oven. Strips were then washed and stained in the GeneAtlas robotic system (Affymetrix) using the GeneAtlas Hybridization, Wash, and Stain Kit for WT Array Strips (Affymetrix). Finally, strips were scanned using the GeneAtlas Imaging System (Affymetrix) generating the raw cel files. Raw intensity values in the cel files, were background corrected, log2 transformed and then quantile normalized by the software Expression Console (Affymetrix) using the Robust Multi-array Average (RMA) algorithm. Finally, comparison of the relative log expression signal (RLE) by box-plot and Principal Component Analysis (PCA) was performed between samples (Fig. 2a-b). PCA analysis show that infected treatments can be distinguished from controls, with virulent and attenuated groups separating further than the saprophyte bacteria. Next, statistical analysis was performed in the TAC software (Affymetrix) by ANOVA and p-values were corrected for multiple hypothesis testing using the Benjamini-Hochberg procedure (fold change 7 2, FDR-corrected p o 0.01).

Identification of differentially expressed genes
Following normalization, CHP files were utilized to identify differentially expressed genes using the software Transcriptome Analysis Console (Affymetrix). Infection by L. interrogans (virulent and attenuated bacteria) caused statistically significant changes in gene expression in more than 800 genes, when compared to non-infected control cells (Fig. 3a-b). Saprophyte L. biflexa modulated 200 genes when compared to control (Fig. 3c). Hierarchical clustering and differences in average signal  (Fig. 3d). Canonical pathways of the differentially expressed genes were identified using the Ingenuity Pathway Analyses (IPA®) software.

Validation of transcriptome results by qRT-PCR
For validation of microarray data between treatments, M-MLV reverse transcriptase (Thermo Fisher Scientific) and QuantiTect SYBR® Green (Qiagen) were used for preparation of cDNA and qPCR, respectively, according to manufacturer´s instructions. PCR was performed in an MxPRO3005 realtime PCR machine (Agilent, Santa Clara, CA, USA). Standard curves were used for all genes at each individual run, and the expression of candidate genes was evaluated as a ratio to the housekeeping control, which was chosen on previous comparative analysis on these samples, between three housekeeping genes. All primers were designed to span at least one intron, to avoid repeat regions and similarities to other non-specific genomic regions. Murine sequence, available on the University of California, Santa Cruz (UCSC) Genome Browser, was employed for primer design using the Primer3 program [2].

Statistical analysis
Differential expression of each gene was determined by ANOVA with two criteria, a fold change of 7 2 comparing all infected groups to the non-infected control and filtered by FDR-corrected p-value o 0.05 Real time PCR data was analyzed using least-squares analysis of variance and the general linear model procedures of SAS (SAS Institute, Cary, NC, USA; p o 0.01). Comparison of means was done using Duncan's multiple range test, and significance was set at p o 0.05.