Challenges of modeling pristane‐induced lupus in non‐human primates

Abstract

A lupus‐like condition induced by intraperitoneal administration of pristane (2,6,10,14‐tetramethylpentadecane) in mice is widely used as a model of systemic lupus erythematosus (SLE). Due to their phylogenetic distance from humans, murine models are not always suitable tool for studying the specific activity of therapeutic agents and the pathogenesis of SLE. In order to overcome species‐specific limitations of murine models, this approach was tested in non‐human primates—cynomolgus monkeys (Macaca fascicularis). Two intraperitoneal injections at a dose of 3.5 mL/kg, administered at weeks 1 and 23, recapitulated SLE features, including: production of antinuclear autoantibodies (ANA), membranoproliferative glomerulonephritis with immune complex (IC) deposition in the glomeruli. However, from week 27 five of eight pristane‐treated monkeys developed progressive respiratory failure. Two of these died at week 28 and the remaining were euthanized at week 32. The histology of the monkey lungs suggested exogenous lipoid pneumonia. Thus, while pristane induced serological autoimmunity and characteristic renal manifestations in Macaca fascicularis, the consequent lipoid pneumonia limited the observation period and prevented comprehensive evaluation of SLE manifestations beyond 32 weeks.

Systemic lupus erythematosus (SLE) in cynomolgus monkeys was modeled by two intraperitoneal injections of pristane at a dose of 3.5 mL/kg, administered at weeks 1 and 23. Serum samples from all monkeys that received pristane tested positive for antinuclear antibodies. Histological examination of the kidneys revealed membranoproliferative glomerulonephritis, and further analysis using immunofluorescent staining of tissue sections showed immune complex deposition in the glomeruli. From week 27, the animals exhibited signs of respiratory failure, and either died or were euthanized between weeks 28 and 32 after the first pristane injection. The histology of the monkey lungs suggested exogenous lipoid pneumonia.

1. INTRODUCTION

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease of unclear etiology with a wide range of clinical manifestations. The systemic nature of the disease stems from two critical immune regulatory defects: the loss of tolerance to nuclear antigens and aberrant activation of the interferon system. To treat immune dysregulation in SLE, both non‐specific anti‐inflammatory and immunosuppressive agents (such as antimalarials, glucocorticoids, and non‐steroidal immunosuppressants) and targeted therapies are used. ¹ , ² Two targeted therapies have been approved by the U.S. Food and Drug Administration for the treatment of SLE: belimumab, a monoclonal antibody against the interferon‐induced B‐cell activating factor, and anifrolumab, a monoclonal antibody that blocks a subunit of the type I IFN (IFN‐I) receptor. ³ , ⁴ Although the development of these drugs represents a major therapeutic breakthrough, primary endpoints were not achieved in 40%–50% of SLE patients enrolled in clinical trials. ⁵ , ⁶ , ⁷ This underscores the need for new treatment approaches and further investigation into the pathogenesis of the disease.

To investigate the genetic and cellular mechanisms of SLE and to screen new therapeutic approaches, various murine models have been developed. ⁸ , ⁹ Unfortunately, due to the phylogenetic distance between mice and humans, some key pathogenesis features cannot be fully replicated in these models. Moreover, certain targeted therapies may not bind to murine molecular targets. For example, the intraperitoneal pristane‐induced SLE model reproduces several disease manifestations, including the production of a broad spectrum of antinuclear antibodies, IC‐mediated glomerulonephritis, proteinuria, arthritis, and increased IFN‐I production. ¹⁰ , ¹¹ , ¹² , ¹³ However, in humans, plasmacytoid dendritic cells (pDCs) are the primary source of IFN‐I, whereas in pristane‐induced murine SLE, disease development depends on IFN‐I produced by peritoneal monocytes. Depletion of pDCs in mice has minimal impact on IFN‐I levels or the expression of interferon‐stimulated genes. ¹⁴ , ¹⁵ , ¹⁶

Successful establishing of lupus‐associated model with systemic lupus syndrome in non‐human primates (Macaca fascicularis) via two intraperitoneal injections of pristane has previously been reported. ¹⁷ That study demonstrated the development of clinical and immunological manifestations of the condition in experimental animals fulfilled the diagnostic criteria for human SLE.

This article reports challenges encountered in modeling SLE in non‐human primates using intraperitoneal pristane administration, specifically the development of exogenous lipoid pneumonia in all experimental animals that received pristane.

2. METHODS

2.1. Animals and experimental housing conditions

The study was approved by the Bioethics Commission of The Federal State Budgetary Scientific Institution “Research Institute of Medical Primatology” (RIMP), Sochi, Russia (approval ID 101‐30.09.2022).

Female cynomolgus monkeys (Macaca fascicularis, n = 9), aged 4–18 years, were obtained from the RIMP. Animals were housed individually in indoor cages equipped with automatic waterers and feed bins at the RIMP facility and received a balanced diet of dry granulated feed supplemented with eggs, dried and fresh fruits (banana, apple, orange), bread, and porridge. The animals were given ad libitum access to water from the central water supply through a stationary watering system. Housing conditions were maintained at 21°C–28°C, 40%–70% relative humidity, under a natural light cycle.

2.2. Induction of condition

Eight animals received intraperitoneal injections of pristane (Pristane, Sigma, purity ≥98%) at a dose of 3.5 mL/kg under sedation with Zoletil® (0.05 mL/kg, IM) and 2% xylazine (0.05 mL/kg, IM) on weeks 1 and 23 of the experiment. Following injection, the abdominal area of each monkey was gently massaged to ensure even distribution of the substance within the peritoneal cavity, as previously described. ¹⁷

One animal (ID 35910) did not receive pristane and served as an untreated control.

2.3. Clinical examination

Animals were monitored daily for clinical changes. The following parameters were evaluated during examination: body posture, condition of the eyes and nose, stool, appetite, respiration pattern, coat condition. Special attention was paid to signs indicative of lupus‐like disease, including malar rash (butterfly‐shaped erythema) over the cheekbones, skin lesions, scabs, generalized or focal alopecia, joint swelling, and neurological and/or cognitive impairments. ¹⁸

2.4. Sampling

Blood samples were collected biweekly from the femoral vein beginning at week 16. For the assessment of autoantibodies level and blood urea nitrogen (BUN) concentration, blood was collected in VACUETTE® CAT serum clot activator tubes. Serum was separated and stored at −20 °C until analysis. For coagulation tests, blood was taken into S‐Monovette® sodium citrate tubes and obtained plasma was analyzed on the same day. Urine was collected biweekly in the morning directly from the cage tray into Falcon‐type tubes.

2.5. Autoantibody concentration measurement

Serum ANA and anti‐double‐stranded DNA antibodies (anti‐dsDNA) levels were measured by ELISA with the commercially available Monkey Anti‐Nuclear Antibody ELISA Kit and the Monkey Anti‐Double Stranded DNA ELISA Kit (MyBioSource, USA), according to the manufacturer's instructions. Absorbance was read at 450 nm on a TECAN Infinite 200 Pro microplate reader.

2.6. Blood urea nitrogen concentration measurement, urinalysis

Serum urea concentration was determined using a kinetic method on a BioChem FC‐360 biochemistry analyzer. The measurement was performed using a reagent kit from Human GmbH (Germany; catalog number: 10521). To calculate the BUN level, the urea concentration was multiplied by a conversion factor of 0.47.

Urinalysis was performed on a DocUReader 2 PRO urine analyzer using Labstrip U11 Plus test strips from the same manufacturer.

2.7. Coagulation analysis

Hemostasis parameters (prothrombin time (PT) and activated partial thromboplastin time (aPTT)) were measured using a TS4000 Plus coagulation analyzer. Reagent kits (RPA Renam, Russia) were used to measure the evaluated parameters.

2.8. Euthanasia

The animals were euthanized by intravenous administration of 5% Anestofol® (5 mL), 2% xylazine (0.10 mL/kg) and Zoletil® (0.05 mL/kg) at week 32.

2.9. Necropsy and histological examination

Necropsy was performed with complete organ evisceration. The organs of the skull, thoracic and abdominal cavities, pelvic organs were examined.

For histological examination, organs and tissues were fixed in 10% neutral buffered formalin. Afterwards, the material was dehydrated, soaked in paraffin, and 3–4 μm thick sections were made from paraffin blocks. The sections were stained with hematoxylin and eosin, Congo red, Van Gieson stain, and periodic acid‐Schiff (PAS) reagent. Tissue specimens were examined using a light microscope Olympus CX41.

2.10. Immunofluorescent staining

To visualize ICs, kidney tissue sections were treated in citrate buffer (pH 6.0; Abcam, UK) for 10 min at 95°C, washed and incubated with FITC‐conjugated goat F(ab′)₂ anti‐human IgG antibodies (Invitrogen, USA) for 60 min at room temperature. After two additional washes in PBS, the sections were counterstained with phalloidin conjugated to Alexa Fluor 648 dye (Invitrogen, USA) and the cell nuclei were stained using Hoechst 33258 (Invitrogen, USA). A series of microimages was acquired using an IN Cell Analyzer 6000.

3. RESULTS

3.1. Clinical examination

Throughout the experiment, no clinical signs suggestive of lupus‐like disease were observed in the monkeys. No butterfly‐shaped erythema on the skin over the cheekbones and no scabs were observed. The animals' coats retained a healthy appearance, with no signs of generalized or focal alopecia. The soft tissues around the joints showed no signs of swelling. Neurological status remained unremarkable throughout the study period. However, between weeks 27 and 30 following the initial intraperitoneal administration of pristane, five out of eight experimental animals displayed clinical signs, including decreased appetite, pale mucous membranes, dyspnea, increased respiratory rate, and reduced physical activity. As pulmonary insufficiency progressed, two animals in the experimental group died at week 28. Three others were euthanized on humane grounds at week 32 of the study. Detailed information for each experimental animal is presented in Table 1.

TABLE 1.

Time of onset and duration of clinical abnormalities observed in the cynomolgus monkeys following intraperitoneal pristane administration at dose of 3.5 mL/kg on weeks 1 and 23.

Animal number	Onset time, weeks	Death/euthanasia, weeks
36831	27	28
45349	28	32
38624	30	32
43734	30	32
37220	27	28

3.2. Autoantibodies

Sera from all pristane‐treated monkeys were positive for ANA from week 18 to week 32 (Figure 1A). In most animals, ANA levels remained within the range of 48–154 ng/mL throughout the experiment, with no noticeable upward trend over time. Monkey No. 44471 showed consistently higher ANA levels, ranging from 308 to 728 ng/mL, than the other pristane‐treated animals. The intact control (monkey No. 35910) stayed below the detection limit throughout.

FIGURE 1.

Serum autoantibody levels in cynomolgus monkeys after intraperitoneal pristane administration (3.5 mL/kg on weeks 1 and 23). (A) Antinuclear antibody (ANA) titres. (B) Anti‐double‐stranded DNA (anti‐dsDNA) titres. Monkey 35 910 served as an intact, non‐induced control.

Circulating anti‐dsDNA were detected in all experimental animals, including the monkey that did not receive pristane, beginning at week 16 of the experiment, with values ranging from 329 to 1958 ng/mL (Figure 1B).

3.3. Coagulation system and hematological parameters

Throughout the study, the changes in coagulation parameters (aPTT and PT) in pristane‐treated monkeys were comparable to those observed in the intact control.

According to the complete blood count results, anemia was detected in three pristane‐treated monkeys, characterized by decreased hemoglobin level, erythrocyte count, and hematocrit (see Table 2). Monkey No. 36831 exhibited elevated platelet counts starting from week 26 (PLT = 1077 − 1233 × 10⁹/L).

TABLE 2.

Values of hematological parameters in the cynomolgus monkeys that developed anemia after pristane administration (values of the first measurement/lowest values during the observation period).

Animal number	HGB (g/L)	HCT (%)	RBC (1012/dL)	Time of anemia onset/weeks
45349	124/93	41.6/31.2	6.2/4.7	28
43734	121/96	39.3/32.2	5.4/4.5	22
36831	118/85	39.9/31.5	6.5/5.6	22

Abbreviations: HCT, hematocrit; HGB, hemoglobin; RBC, red blood cell.

3.4. Renal manifestations (BUN, kidney function and histopathology, IC deposition)

During the observation period, only two monkeys that received pristane showed an increase in BUN levels compared to earlier time points. In monkey No. 38624, BUN remained at 2.7 ± 0.4 mmol/L for 8 weeks from week 16 to week 24 of the experiment, then gradually increased, reaching a peak at week 32 (14.1 mmol/L). In monkey No. 37220, BUN was at 1.9 ± 0.3 mmol/L from week 16 to week 26, with an increase observed at week 28; further assessment of changes was not possible due to the animal's death at week 28 (Figure 2).

FIGURE 2.

Serum blood‐urea‐nitrogen (BUN) concentrations in individual cynomolgus monkeys after intraperitoneal pristane administration. BUN levels were measured biweekly started at week 16 after the first pristane injection. Two monkeys that received pristane (3.5 mL/kg on weeks 1 and 23) displayed a marked elevation in BUN relative to their own earlier values, whereas the remaining animals maintained stable concentrations throughout the study period. Individual data are shown.

The urinalysis did not reveal any kidney function abnormalities in the experimental animals: the assessed parameters remained within normal ranges throughout the experiment (data not shown).

Histological examination of the kidneys in all experimental animals that received pristane revealed membranoproliferative glomerulonephritis (Figure 3A). Immunofluorescent staining of the tissue sections identified the deposition of ICs in the kidney glomeruli in the monkeys that received pristane (Figure 3B).

FIGURE 3.

Renal pathological changes in the cynomolgus monkey following intraperitoneal pristane administration. (A) Representative images of PAS‐stained kidneys sections. All experimental animals that received pristane revealed membranoproliferative glomerulonephritis. Scale bar: 50 μm (B) Immune‐complex deposition (green fluorescence marks) in the glomeruli, indicating an autoimmune nature of the glomerulonephritis. Sections were stained by FITC‐conjugated goat F(ab′)₂ anti‐human IgG. Scale bar: 100 μm.

3.5. Pathological examination

The pathological examination was performed for all animals that received pristane. For monkeys No. 36831 and No. 37220, the examination was conducted at 28 weeks after the first pristane injection, and for the remaining animals, at week 32.

During the necropsy and assessment of abdominal organs, all animals had pronounced adhesions at the site of pristane injection, involving the large and small intestines, stomach, pancreas, parietal and visceral peritoneum, greater and lesser omentum, liver, and the abdominal part of the diaphragm, accompanied by parietal granulomatosis. All animals exhibited varying degrees of generalized lymphadenopathy in the thoracic, abdominal, and pelvic cavities. Most of the lymph nodes had a whitish or yellowish color with dense consistency. In some cases, cystic cavities were observed at sites of immune system tissue, with some completely replacing the tissue of the lymph nodes. Notably, in all animals, lung tissue was dense, heavy, with reduced airiness, gray or gray‐pink, with focal hemorrhages or congested blood vessels under the visceral pleura.

Histological examination of lung tissue in all animals revealed numerous foci of granulomatous inflammation, represented by lipogranulomas at different stages of development. In most foci of productive inflammation, an amorphous acellular substance with single vacuoles was observed at the center, where necrotized cells had been (Figure 4A). Perifocal inflammation was represented by macrophages with vacuolated cytoplasm, fibroblasts, a few lymphocytes, and foreign body giant cells. In most cases, lipogranulomas were confluent, leading to changes in the organ architecture. Inflammation was accompanied by fibrosis, deformation of the bronchoalveolar tree, and secondary bronchopneumonia (in monkeys No. 38624 and No. 44471). In some fields of view, the bronchi were cystically dilated, and stagnation of mucous secretion was noted.

FIGURE 4.

Effect of pristane administration on the lungs of the cynomolgus monkeys. (A) Representative images of hematoxylin‐eosin‐stained lungs. Numerous foci of granulomatous inflammation, represented by lipogranulomas at different stages of development, were revealed. Scale bar: 50 μm (B) Representative images of Van Gieson‐stained lungs. Massive pneumofibrosis was observed in the lungs of animals showing clinical signs of respiratory failure, with restructuring of lung tissue and compensatory expansion of the lobular and terminal bronchioles. Scale bar: 100 μm.

In animals showing clinical signs of respiratory failure (Nos 36831, 37220, 45349, 43734, 38624), massive pneumofibrosis was observed in the lungs, with restructuring of lung tissue and compensatory expansion of the lobular and terminal bronchioles (Figure 4B). The epithelium appeared cuboidal, and the blood vessels were unevenly filled with blood.

Additional staining methods did not reveal the presence of amyloid, calcium salts, or mucin (data not shown).

4. DISCUSSION

Our work focused on modeling SLE in non‐human primates using pristane. Pristane is a natural isoprenoid alkane and a component of mineral oil. A lupus‐like condition induced by intraperitoneal administration of pristane is widely used as a model of SLE, as it reproduces many aspects of the human disease.

Following pristane administration, animals develop a chronic inflammatory response, with lipogranulomas forming in the abdominal cavity. Apoptosis of cells induced by pristane provides an autoantigenic substrate for the breakdown of immune tolerance, leading to immune dysfunction associated with the overproduction of IFN‐I, which in turn results in the development of immune system autoaggression. ¹⁹ , ²⁰ The disease pathogenesis includes the production of autoantibodies, the development of IC‐mediated glomerulonephritis, anemia, hemorrhagic pulmonary capillaritis, and arthritis. ¹³ , ²¹

A report has been published on the induction of a lupus‐like condition in cynomolgus monkeys (Macaca fascicularis) following two intraperitoneal injections of pristane at a dose of 3.5 mL/kg. In that study, autoantibodies were detected at week 27 after the first pristane injection, followed by clinical signs such as malar rash, alopecia, skin lesions, arthrocele, and arthritis. The timing of the appearance of clinical signs corresponded to the time when autoantibodies began to be detected. Hematologic abnormalities (↑aPTT, ↑PT) and renal function changes emerged from week 30. ¹⁷

The aim of our study was to induce a lupus‐like condition in monkeys for further evaluation of the specific activity of therapeutic agents. The dose and route of pristane administration were selected for implementation in accordance with the protocol outlined in the report described above, ¹⁷ with pristane administration occurring at weeks 1 and 23. From week 18, the serum of all pristane‐treated monkeys tested positive for ANA, whereas ANA were not detected in the serum of the intact control. However, the ANA levels in most of the animals remained relatively low throughout the experiment, and an increase in ANA levels over time was not observed. Circulating anti‐dsDNA were detected in all experimental animals, including the monkey that did not receive pristane. Throughout the experiment, no clinical signs suggestive of lupus‐like disease were observed in the monkeys. Changes over time in coagulation system parameters were comparable to those of the intact control. Clinical blood tests revealed anemia in three monkeys that received pristane, with a decrease in hemoglobin, hematocrit, and red blood cell count observed from week 22 (2 monkeys) and 28 (1 monkey) of the experiment. One of these animals showed elevated platelet counts (thrombocytosis) starting from week 26.

During the experiment, an increase in BUN levels was observed only in two animals that received pristane. Urinalysis did not detect any kidney dysfunction: the urine parameters were normal throughout the experiment, including the absence of protein and erythrocytes in the urine. However, histological examination of the kidneys from all experimental animals that received pristane revealed membranoproliferative glomerulonephritis, and immunofluorescence staining of kidney sections identified IC deposits in the glomeruli, suggesting an autoimmune cause of the glomerulonephritis. Table 3 summarizes information on the presence/absence of clinical manifestations characteristic of SLE in each experimental animal.

TABLE 3.

Clinical manifestations of SLE observed in the cynomolgus monkeys following intraperitoneal pristane administration.

Animal number	ANA	Renal (BUN)	Renal (glomerulonephritis)	Hematologic (anemia)	Clinical signs	Coagulometric changes
45349	+	−	+	+	−	−
38624	+	+	+	−	−	−
43734	+	−	+	+	−	−
36831	+	−	+	+	−	−
37220	+	−	+	−	−	−
44471	++	−	+	−	−	−
43360	+	−	+	−	−	−
36781	+	−	+	−	−	−
35910 (Intact control)	−	−	−	−	−	−

Note: Minus (−) means the absence of a manifestation, plus (+) means the presence of a manifestation, double plus (++) means strong expression of a manifestation.

Abbreviations: ANA, antinuclear antibodies in serum; BUN, the blood urea nitrogen.

From week 27 of the experiment, five monkeys that were administered with pristane began to show abnormalities in clinical examination that were not related to clinical signs of SLE. The clinical presentation was similar in the animals: decreased appetite, pale mucous membranes, dyspnea, increased respiratory rate and decreased motor activity. Respiratory failure increased over time, two monkeys died at week 28 of the experiment, the remaining monkeys with signs of respiratory failure were euthanized for humane reasons at week 32 of the experiment.

Previously, there were no reports of monkey deaths associated with impaired lung function when inducing a lupus‐like condition with pristane. During the histological examination of the lungs of monkeys receiving pristane, we observed a large number of foci of granulomatous inflammation, represented by lipogranulomas at various stages of development. An amorphous acellular substance with single vacuoles was observed at the center of lipogranulomas, where necrotized cells had been. Perifocal inflammation was represented by macrophages with vacuolated cytoplasm, fibroblasts, a few lymphocytes, and foreign body giant cells. In most cases, lipogranulomas were confluent, leading to changes in the organ architecture. Inflammation was accompanied by fibrosis. In animals showing clinical signs of respiratory failure, massive pneumofibrosis was observed in the lungs, with restructuring of lung tissue and compensatory expansion of the lobular and terminal bronchioles. In general, the histology of the monkey lungs suggested exogenous lipoid pneumonia.

5. CONCLUSION

In this article, we report the challenges encountered in modeling SLE in non‐human primates—cynomolgus monkeys (Macaca fascicularis) using intraperitoneal administration of pristane. The development of exogenous lipoid pneumonia following the administration of pristane aggravates the clinical condition of animals and makes it difficult, and in some cases impossible, to assess the clinical symptoms and laboratory markers of SLE.

AUTHOR CONTRIBUTIONS

Elena Vladimirovna Vinogradova: Data curation; formal analysis; project administration; visualization; writing – original draft. Maria Alexandrovna Varavko: Investigation; writing – original draft; writing – review and editing. Yakov Yuryevich Ustyugov: Conceptualization; supervision; writing – review and editing. Alexey Alexandrovich Aleksandrov: Conceptualization; supervision; writing – review and editing. Dmitry Alexandrovich Khochenkov: Investigation. Yulia Nikolaevna Linkova: Writing – review and editing.

FUNDING INFORMATION

This research received no specific grant from any funding agency.

CONFLICT OF INTEREST STATEMENT

The authors have no conflict of interest to declare.

ETHICS STATEMENT

The study was approved by the Bioethics Commission of The Federal State Budgetary Scientific Institution “Research Institute of Medical Primatology”, Russia (approval ID 101‐30.09.2022).