All experimental protocols utilized in this study comply with the Czech Government Requirements under the Policy of Animal Protection Law (No. 246/1992) and with the regulations of the Ministry of Agriculture of the Czech Republic (No. 207/2004), which are in agreement with all relevant European Union guidelines for work with animals and were approved by the Institutional Animal Care Committee of the Institute of Molecular Genetics AS CR and by Departmental Expert Committee for the Approval of Projects of Experiments on Animals of the Academy of Sciences of the Czech Republic (permissions Nr. 190/2010; 232/2012).

Leishmania major LV 561 (MHOM/IL/67/LRCL 137 JERICHO II) was maintained in rump lesions of BALB/c females. Amastigotes were transformed to promastigotes using SNB-9 (35). 10⁷ promastigotes from the passage two cultivated for 6 days were inoculated in 50 µl sterile saline s.c. into mouse rump (36). Control uninfected mice were injected by 50 µl sterile saline.

The size of the skin lesions was measured every week using the Profi LCD Electronic Digital Caliper Messschieber Schieblehre Messer (Shenzhen Xtension Technology Co., Ltd. Guangdong, China), which has accuracy 0.02 mm. The mice were killed 8 weeks after inoculation. Skin, spleen, liver, and inguinal lymph nodes were collected for later analysis. Preparation of skin samples: approximately 3 mm of border skin surrounding lesion was taken. Hair was removed with scissors. A half of each skin sample was snap-frozen in liquid nitrogen for further RNA and DNA isolations. Another half was fixed in 4% formaldehyde for further paraffin embedding and immunohistochemical analysis. Samples from uninfected mice were obtained from the same rump area and used as a negative control.

Parasite load was measured in DNA from frozen skin and spleen samples using PCR-ELISA according to the previously published protocol (37). Briefly, total DNA was isolated using a TRI reagent (Molecular Research Center, Cincinnati, OH, USA) standard procedure (https://www.mrcgene.com/wp-content/uploads/2014/06/TRI-LSMarch2017.pdf) or a modified proteinase K procedure (37). For PCR, two primers (digoxigenin-labeled F 5′-ATT TTA CAC CAA CCC CCA GTT-3′ and biotin-labeled R 5′-GTG GGG GAG GGG CGT TCT-3′; VBC Genomics Biosciences Research, Austria) were used for amplification of the 120-bp conservative region of the kinetoplast minicircle of Leishmania parasite, and 50 ng of extracted DNA was used per each PCR reaction. For a positive control, 20 ng of L. major DNA per reaction was amplified as a highest concentration of standard. A 33-cycle (expression experiments) or 26-cycle (immunostaining experiments) PCR reaction was used for quantification of parasites. Under these conditions, the amount of PCR product is linearly proportional to number of parasites (37). PCR product was measured by the modified ELISA (Pharmingen, San Diego, CA, USA). Concentration of Leishmania DNA was determined using the ELISA Reader Tecan and the curve fitter program KIM-E (Schoeller Pharma, Prague, Czech Republic) with least squares-based linear regression analysis.

Mouse spleens, skins, liver, and inguinal lymph nodes were snapped frozen by liquid nitrogen immediately after dissection and stored at −80°C until total RNA extraction. At the time of RNA isolation tissue were homogenized in TRI Reagent (Sigma-Aldrich, Inc., St. Louis, MO, USA) using Polytron PT 2100 homogenizer (Kinematica Ag, Luzern, Switzerland) and immediately followed by total RNA isolation according to the manufacturer’s protocol. RNA concentration was measured with Nanodrop (NanoDrop Technologies, LLC, Wilmington, DL), and quality of RNA was estimated also using Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA, USA). The isolated RNA was stored at −80°C.

One microgram of total RNA was diluted in 8 µl of sterile RNase- and DNase-free water, was treated with 1 µl DNase I (1 U/μl) and 1 µl DNase I reaction buffer (Promega Corporation, Madison, WI, USA), and used for subsequent reverse transcription. Single-strand cDNA was prepared from total RNA using Promega first-strand synthesis method. DNase I-treated RNA was incubated for 10 minutes at 65°C, then cooled quickly on ice for 5 minutes, and then treated with 1 µl DNase I stop solution (Promega Corporation, Madison, WI, USA). For the next step, a mixture containing 1 µl of random hexamers primers (100 ng/1 µl) (Invitrogen, Carlsbad, CA, USA), 5 µl (50 ng/µl) of dNTP mix (Invitrogen, Carlsbad, CA, USA), 5 µl of the reaction buffer (Promega Corporation, Madison, WI, USA), 2.5 µl of RNase/DNase-free water (Invitrogen, Carlsbad, CA, USA), and 0.5 µl of M-MLV Reverse Transcriptase RNAase H Minus Point Mutant (100 U/1 μl) (Promega Corporation, Madison, WI, USA) was added and followed by 60 minutes at 37°C. Single-strand cDNA was kept at −80°C until RT-PCR analysis. Real-time PCR was performed using a BioRad iQ iCycler Detection System (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Primers were designed using Roche Universal ProbeLibrary, ProbeFinder version 2.45 for mouse (Gbp2b/Gbp1-F AAACCAGGAGGCTACTACCTTTTT, Gbp2b/Gbp1-R GTATTTTCTCAGCATCACTTCAGC; Gbp5-F TTCACCCAATCTAAGACCAAGAC, Gbp5-R AGCACCAGGCTTTCTAGACG; Gapdh-F AGAACATCATCCCTGCATCC, Gapdh-R ACATTGGGGGTAGGAACAC). Reaction was performed in total volume of 25 µl, including 12.5 µl of 2× SYBR Green Supermix (Bio-Rad Laboratories, Inc., Hercules, CA, USA), 1 µl of each primer of Gbp2b/Gbp1 and Gbp5 genes (final concentration 6.6 µM), 7.5 µl of water (Invitrogen, Carlsbad, CA, USA), and 3 µl of the cDNA template. Six different samples from each experimental group were used, and all samples were tested in triplets. The average Ct values (cycle threshold) were used for quantification, and the relative amount of each mRNA was normalized to the housekeeping gene, Gapdh mRNA. Using the delta Ct value, relative expression was calculated [ratio (reference/target) = 2 Ct (reference) − Ct (target)] × 10,000. All experiments included negative controls containing water instead of cDNA.

DNA was isolated from tails using a standard proteinase procedure. Strains O20, B10, B10.O20, OcB-9, and OcB-43 were genotyped using microsatellite markers D3Mit160 (size of B10 allele 137 bp, size of O20 allele 127 bp) and D3Mit17 (B10 allele 200 bp, O20 allele 174 bp) (Generi Biotech, Hradec Králové, Czech Republic): The DNA genotyping by PCR was performed as described elsewhere (38).

After deparaffinization and rehydration, the 3 µm thick slices of skin tissue were 15 minutes heat induced in Tris-EDTA buffer (10 mM Tris, 1 mM EDTA, pH 8.5) for antigen retrieval. For fluorescent labeling of Leishmania parasite was used anti-Leishmania lipophosphoglycan mouse monoclonal antibody (cat. no. CLP003A; Cedarlane, Hornby, Canada) and TRITC-labeled IgM (115-025-020; Jackson ImmunoResearch, West Grove, PA) all diluted 1:500. Gbp2b/Gbp1 protein was stained with rabbit anti-Gbp1 Polyclonal antibody (PA5-23509; Thermo Fisher Scientific, Rockford, IL, USA) diluted 1:100 and anti-rabbit-AlexaFluor-647 (cat. no. 711-605-152; Jackson ImmunoResearch, West Grove, PA) diluted 1:500. Nuclei of the cells were stained with bisBenzimide H33258 (Sigma-Aldrich, St. Louis, MO, USA) 10 mg per 1 ml diluted 1:1,000. Images were captured with microscope Leica DM6000 objective HCX PL Apo 40×/0.75 PH2 and color camera Leica DFC490. Evaluation of images was done with Fiji ImageJ 1.51n. 10 fields (320.66 × 239.57 µM) from each mouse were analyzed.

The differences among strains within each of the two groups in expression of Gbp2b/Gbp1 and Gbp5 and the differences between uninfected and infected mice were evaluated by Mann–Whitney test using the program Statistica for Windows 12.0 (StatSoft, Inc., Tulsa, OK, USA). The results were corrected for multiple testing by Bonferroni correction. The correction factor was 10× both for intragroup differences and differences between infected and uninfected mice of the same strain.

We observed strong genetic influence on mRNA levels of tested Gbps. We have examined expression of Gbp2b/Gbp1 (Figure 1) and Gbp5 (Figure 2) in skin, inguinal lymph nodes, spleen, and liver of uninfected mice belonging to two genetically different series of strains—CcS/Dem (BALB/c, STS, CcS-5, CcS-16, CcS-20) and OcB/Dem (O20, B10, B10.O20, OcB-9, OcB-43). We have compared expression in parental strains BALB/c and STS with the strains of CcS/Dem series, and expression in parental strains O20 and B10 with the strains of OcB/Dem series (Figures 1 and 2), as well as expression of the strains within CcS/Dem and OcB/Dem series in skin (Tables 1A,C), lymph nodes (Tables 2A,C), spleen (Tables 3A,C), and liver (Tables 4A,C).

Expression of Gbp2b/Gbp1 in background strain BALB/c and donor strain STS in skin (Figure 1A; Table 1A) does not differ, whereas strains CcS-5, CcS-16, and CcS-20, each carrying a different set of 12.5% genes of STS on BALB/c background, exhibit higher expression than either parent (Figure 1A; Table 1A). Expression of Gbp2b/Gbp1 in parental strains of OcB/Dem series O20 and B10 in skin differed (P = 0.0047); strains B10.O20, OcB-9, and OcB-43 exceeded in Gbp2b/Gbp1 expression of the parental strain B10 but not O20 (Figure 1A; Table 1A).

We have observed differences in the expression of Gbp2b/Gbp1 among mouse strains also in other tested organs (Figures 1B–D; Tables 2A, 3A, and 4A). Strains OcB-43 and OcB-9 differed in the expression of Gbp2b/Gbp1 in lymph nodes (Table 2A), CcS-16 exhibited lower expression than STS in spleen (Figure 2C; Table 3A), B10 and B10.O20 exhibited lowest expression in spleen and differed from strains O20, OcB-9, and OcB-43, CcS-5 exhibited lower expression than CcS-16 in liver (Figure 1D; Table 4A); however, expression in none of the CcS or OcB strains exceeded the range of expression in both parental strains.

Expression of Gbp5 in skin did not differ in parental strains of CcS/Dem series BALB/c and STS (Figure 2A; Table 1C), and expression of Gbp5 in CcS-20 exceeded the expression of both parental strains (Figure 2A; Table 1C). There was no difference in Gbp5 expression among strains of OcB/Dem series (Table 1C).

We did not find any significant differences in expression of Gbp5 among the strains of both CcS/Dem and OcB/Dem series in the lymph nodes, spleen, and liver; none of the strains in CcS/Dem or in OcB/Dem series differed from either parent (Figures 2B–D; Tables 2C, 3C, and 4C).

In susceptible mice, pathology started as a nodule at the site of L. major infection appearing between weeks 2 and 4, which progressed in susceptible strains into a skin lesion (Figure 3A) (34). Strains BALB/c and CcS-16 are highly susceptible and develop large skin lesions (Figures 3A,B), B10.O20 develops moderate skin lesions (Figures 3A,C), CcS-20 is intermediate (Figures 3A,D) (34), and STS, CcS-5, O20, B10, OcB-9, and OcB-43 are resistant with no or minimal skin lesions (Figure 3A). All tested strains contain parasites in skin (Figure 3D) and spleen (Figure 3E), although the parasite load in resistant strains STS, CcS-5 and O20 (skin and spleen), and OcB-9 (spleen) is low.

Infection increased the expression of Gbp2b/Gbp1 and Gbp5 in organs of tested mice, the highest increase was observed in skin (Figures 4–7). All tested strains except CcS-5 and OcB-9 exhibited significantly higher expression of Gbp2b/Gbp1 and Gbp5 in skin after infection, irrespective of their susceptibility or resistance status (Figure 4A). Similarly as in uninfected mice, levels of Gbp2b/Gbp1 mRNA in CcS-16 and CcS-20 exceeded those in both parental strains BALB/c and STS (Figure S1A in Supplementary Material; Table 1B); Gbp5 expression in infected CcS-20 also exceeded that in both BALB/c and STS (Figure S2A in Supplementary Material; Table 1D).

Infection also induced increase of Gbp2b/Gbp1 in inguinal lymph nodes of all strains except BALB/c and CcS-20, the highest expression was observed in CcS-5 (Figure 5A), which differed from all tested strains except STS (Figure S1B in Supplementary Material; Table 2B), but only increase of expression of B10.O20 and OcB-43 was significant after correction for multiple testing; we did not observe significant increase of Gbp5 mRNA in lymph nodes (Figure 5B).

Four strains (BALB/c, STS, CcS-5, and CcS-16) show significantly increased expression of Gbp2b/Gbp1 in spleen (Figure 6A). Expression of Gbp5 was increased in CcS-5 (Figure 6B).

In liver, infection induced significant increases of Gbp2b/Gbp1 mRNA in strains of CcS/Dem series, CcS-5, and CcS-16 (Figure 7A). Level of Gbp2b/Gbp1 mRNA in CcS-16 is highest from all tested strains (Figure S1 in Supplementary Material; Table 4B). Expression of Gbp5 was significantly increased in CcS/Dem strains CcS-5 and CcS-16 and decreased in OcB/Dem strain OcB-43 (Figure 7B; Table 4D).

Expression of Gbp2b/Gbp1 mRNA was highest in skin of infected mice (Figure S1 in Supplementary Material; Figure 4), we have therefore analyzed by immunohistochemistry a presence of Gbp2b/Gbp1 protein in the skin of selected strains BALB/c, STS, CcS-5, CcS-20, and O20 and its relationship to L. major parasite in infected mice. Figure 8 shows the presence of Gbp2b/Gbp1 protein in the skin of uninfected strains. The comparison of position of L. major and Gbp2b/Gbp1 in the skin of chronically infected highly susceptible strain BALB/c showed few Gbp2b/Gbp1 in the vicinity of L. major parasites, but a large part of parasites was free of Gbp2b/Gbp1 (Figure 9A); the comparison of parasite load in the skin of the tested strains is shown in Figure S3 in Supplementary Material. In resistant strains STS (Figure 9B), CcS-5 (Figure 9C), and O20 (Figure 9E) and in intermediate strain CcS-20 (Figure 9D), Gbp2b/Gbp1 co-localized with clusters of parasites (Figures 9B–E) that in some places formed large clusters or long stretches. Gbp2b/Gbp1 either surrounded these clusters (Figures 9B–D) or formed stretches consisting of L. major parasites and Gbp2b/Gbp1 (Figures 9C,E). The tightest co-localization was observed in strains CcS-20 (Figure 9D) and O20 (Figure 9E).

Tested strains exhibited genetic differences in Gbps expression both before and after L. major infection (Figures 1, 2, 4 and 7; Tables 1–4). Our study extends analysis of genetic influence by Staeheli and coworkers on Gbp2b/Gbp1 expression (39), who injected forty six mouse strains by poly(I);poly(C) in order to induce interferon production and tested their spleen cells for guanylate-binding activity. Tested strains were divided into Gbp2b/Gbp1 inducible and Gbp2b/Gbp1 noninducible groups. BALB/c was in the inducible group, whereas STS, O20, and C57BL/6J belonged to noninducible one (39). Our data confirm strong genetic influence on expression of Gbp2b/Gbp1; however, a direct comparison of outcome of study of Staeheli et al. (39) with our results is impossible due to different experimental designs. They induced Gbp2b/Gbp1 expression by poly(I);poly(C) that is structurally similar to double-stranded RNA present in some viruses, whereas we stimulated Gbp2b/Gbp1 expression by the chronic infection with parasite L. major.

Our data surprisingly showed that in several organs expressions levels of Gbps in recombinant congenic strains were outside the range of their parents. In skin of uninfected mice, expression of Gbp2b/Gbp1 in CcS-5, CcS-16, and CcS-20 exceeded those of both their parents BALB/c and STS (Figure 1A) and expression of Gbp2b/Gbp1 in B10.O20 exceeded expression in parental strain B10 (Figure 1A). Such pattern of inheritance has been considered to be caused by trans-regulatory effects of non-linked or distant genes (40). The differences between parental strains and CcS/Dem strain CcS-20 persist after L. major infection, whereas the differences between expression of parents and CcS-5 and CcS-16 and between parent B10 and the strain B10.O20 disappear after infection (Figure 1A; Figure S1A in Supplementary Material; Tables 1A,B). Expression of Gbp5 in skin of uninfected CcS-20 exceeded level of both parents (Figure 2A; Table 1C) but was significantly higher only than the parental strain STS after 8 weeks of infection (Figure S2A in Supplementary Material; Table 1D). CcS-5 and CcS-16 highly differed in the expression of both Gbp1/Gbp2b and Gbp5 in lymph nodes and liver of infected mice; these strains also differed in expression of Gbp5 in spleen (Tables 2B,D, 3D, and Tables 4B,D).

Comparison of genotypes of the tested strains (33, 41, 42) (this study) in the Gbp cluster on the mouse chromosome 3 (Figure 10) revealed that strains CcS-5, CcS-16, and CcS-20 exhibiting higher expression of Gbp had Gbp genotype identical to that of BALB/c (C). Similarly, highly differing CcS-5 and CcS-16 strains carry the same Gbp allele. The presence of the same allele of Gbp gene cluster on chromosome 3 in strains that differ in other genes suggests that their differences in expression of Gbp2/Gbp1 and/or Gbp5 from one or both parents or from other RC strain are due to regulatory influence of non-Gbp gene(s) of STS origin carried on other genetic segments (trans-regulation). In the OcB/Dem series, B10.O20 carried in Gbp cluster B10 genotype (B), which similarly indicated trans-regulation of expression from O20 genome situated outside Gbp cluster (Table 1A; Figure 10). This trans-regulation can be partly overlaid by other regulatory events appearing after infection. Further genetic studies will be needed to elucidate nature of regulatory events observed in our studies.

The observations of progeny having a phenotype, which is beyond the range of the phenotype of its parents, are not rare. For example, analysis of gene expression from livers in chromosome substitution mouse strains revealed that only 438 of the 4,209 expressed genes were inside the parental range (40). These observations are due to multiple regulatory interactions, which in new combinations of these genes in recombinant congenic or chromosomal substitution strains can lead to the appearance of new phenotypes that exceed their range in parental strains.

We and others have demonstrated that Leishmania parasites are present not only in organs of infected susceptible mice with clinical manifestations of the disease but also in clinically asymptomatic mice of resistant strains (37, 43–46). This is also shown in Figures 3D,E and 9B,C,E. Figures 4–7 show that the expression of Gbp2b/Gbp1 and/or Gbp5 has increased after infection in at least one organ of each of the tested mice, including the resistant ones (STS, CcS-5, O20, B10, OcB-9, and OcB-43), which had no or only minimal and transient clinical symptoms. This strongly suggests that persistent parasites can contribute to the maintenance of protective immunity, which was manifested in our experiments by the increased levels of Gbp2b/Gbp1 and Gbp5 in resistant mice. It was demonstrated previously that this latent infection is controlled by inducible nitric oxide synthase (43) and phagocyte NADPH oxidase (46). It remains to be established, whether defense mechanisms including Gbps that were found to act against other pathogens (16, 23), operate also in Leishmania-infected mammalian host. In defense against M. bovis, Gbp2b/Gbp1 and Gbp7 could promote NADPH oxidase activity after the recruitment of gp91phox and gp22phox components to bacteria vacuoles (23), whereas parasite T. gondii was directly attacked via Gbp supramolecular complexes (16). The observed association (Figure 9) of Gbp2b/Gbp1 with L. major parasites in the skin of resistant and intermediate strains but not the highly susceptible strain BALB/c may suggest a role of this protein in response against the L. major pathogens.

Importantly, persistent parasites, besides stimulating protective immune reactions, can also represent a danger for hosts (45). The increased expression of Gbp2b/Gbp1 and Gbp5 in clinically asymptomatic mice reveals the price exacted from the organism by a dormant infection. This finding deserves attention, because elevated levels of human GBP1 are directly involved in the endothelial dysfunction and the regulation of endothelial progenitor cells activity in patients with the autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus and systemic sclerosis (47). In mice, elevated levels of Gbp3 and Gbp6 were linked with the pathogenesis of atherosclerosis (48). In humans with colorectal cancer, the anti-angiogenic effect of increased levels of GPB1 was beneficial in colorectal carcinoma patients, where it was associated with sustained reduction of intratumoral angiogenic activity and improved cancer-related survival (49).

The immune reactions accompanying persistent Leishmania infection might be very important, because in addition to 12 million people presently suffering from the clinical manifestations of leishmaniasis (50), there are at least 120 million people with asymptomatic infection (45). It needs to be elucidated, whether such clinically asymptomatic people harboring persistent Leishmania parasites are more prone to immune-related diseases.