IL-1 Receptor Antagonist Treatment Aggravates Staphylococcal Septic Arthritis and Sepsis in Mice

Abstract

Background: Interleukin-1 receptor antagonist (IL-1Ra) is the primary therapy against autoinflammatory syndromes with robust efficacy in reducing systemic inflammation and associated organ injury. However, patients receiving IL-1Ra might be at increased risk of acquiring serious infections.

Aims: To study whether IL-1Ra treatment deteriorates Staphylococcus aureus (S. aureus) septic arthritis and sepsis in mice.

Method: NMRI mice were treated with anakinra (IL-1Ra) daily for 7 days before intravenous inoculation with S. aureus strain Newman in both arthritogenic and lethal doses. The clinical course of septic arthritis, histopathological and radiological changes of the joints, as well as the mortality were compared between IL-1Ra treated and control groups.

Results: IL-1Ra treated mice developed more frequent and severe clinical septic arthritis. Also, the frequency of polyarthritis was significantly higher in the mice receiving IL-1Ra therapy. In line with the data from clinical arthritis, both histological and radiological signs of septic arthritis were more pronounced in IL-1Ra treated group compared to controls. Importantly, the mortality of IL-1Ra treated mice was significantly higher than PBS treated controls.

Conclusion: IL-1Ra treatment significantly aggravated S. aureus induced septic arthritis and increased the mortality in these mice.

Fig 1. IL-1Ra therapy aggravated S. aureus arthritis in mice.

NMRI mice inoculated with S. aureus strain Newman (1.1–1.7 × 10⁶ cfu/mouse) were treated with anakinra (IL-1Ra, 0.4mg/mouse, n = 30), or PBS (n = 25) daily starting on day 7 before inoculation with bacteria and continuing until the animals were sacrificed on day 10. The severity (A) and frequency (B) of clinical arthritis in the mice were observed for 10 days post-infection. The frequency of polyarthritis (C) and changes in percentage of body weight (D) of the mice were also registered. The data from 3 independent experiments were pooled. Statistical evaluations were performed using the Mann–Whitney U test and chi-square test. Data are presented as mean±SEM. * = p < 0.05; ** = p < 0.01.

Fig 2. IL-1Ra therapy led to more severe radiologically verified bone destruction in mice.

NMRI mice inoculated with S. aureus strain Newman (1.1–1.7 × 10⁶ cfu/mouse) were treated with anakinra (IL-1Ra, 4mg/mouse, n=30), or PBS (n= 25) daily starting on day 7 before inoculation with bacteria and continuing until the animals were sacrificed on day 10. The accumulative bone destruction scores (A) and frequency of bone destructions (B) from all 4 limbs of NMRI mice by a microCT-scan. Representative CT-scan pictures (C) show intact wrist and knee joints from a NMRI mouse inoculated with S. aureus Newman strain that was treated with PBS and a heavily destructed knee (proximal tibia and head of fibula) and wrist (both distal radius and ulna) joints from a NMRI mice with septic arthritis treated with anakinra. Arrows indicate the bone destructions. Statistical evaluations were performed using the Mann–Whitney U test. Data were presented as mean± SEM. ** = p < 0.01; μCT= Micro Computed Tomography.

Fig 3. IL-1Ra treated mice had more histological signs of septic arthritis.

NMRI mice inoculated with S. aureus Newman strain (1.1–1.7 × 10⁶ cfu/mouse) were treated with anakinra (IL-1 Ra 4mg/mouse, n = 30), or PBS (n = 25) every day starting on day 7 before inoculation with bacteria and continuing until the animals were sacrificed on day 10. (A) Histological evaluation (including the synovitis scores and bone erosion scores) of the joints from all 4 limbs on 10 days after infection. (B) Left panel: A micrograph of histologically intact midfoot joints from a NMRI mouse inoculated with S. aureus strain Newman that was treated with PBS. Right panel: A micrograph of a heavily inflamed midfoot joints with bone and cartilage destruction from a NMRI mouse with septic arthritis treated with anakinra. Hematoxylin/eosin was used for staining; * indicates heavely inflamed synovium; Arrow indicates the bone erosion; S, synovial tissue. Statistical evaluations were performed using the Mann–Whitney U test. Data were presented as scatter dot plot with median; ns = not significant.

Fig 4. IL-1Ra therapy has no effect on either kidney abscesses or S. aureus loads in kidneys and joints.

NMRI mice inoculated with S. aureus strain Newman (1.1–1.7 × 10⁶ cfu/mouse) were treated with anakinra (IL-1Ra, 4mg/mouse, n = 40), or PBS (n = 35) every day starting on day 7 before inoculation with bacteria and continuing until the animals were sacrificed on day 5 and day 10. (A) Abscess scores of the kidneys from the mice sacrificed 10 days after infection and, (B) persistence of S. aureus in kidneys of the mice. The data from 3 independent experiments were pooled, (n = 23–26 mice/group). (C) Persistence of S. aureus in joints including wrists, ankles, and knees of the mice (n = 10 mice/group) sacrificed 5 days after infection. Statistical evaluations were performed using the Mann–Whitney U test. Data were presented as mean± SEM for kidney abscesses or scatter dot plot with median for bacterial load in kidneys and in joints. ns = not significant.

Fig 5. IL-1Ra treatment did not alter whole blood S. aureus killing capacity and macrophage phagocytosis.

Blood and peritoneal macrophages were collected from NMRI mice treated subcutaneously with anakinra (IL-1Ra, 4mg/mouse, n = 5), or PBS (n = 5) for 7 days. (A) Survival of S. aureus Newman strain was analyzed in mouse whole blood, where bacteria were adjusted to 1.0 × 10³ CFU/ml, mixed 1:4 with blood, and incubated at 37°C. (B) Quantitative phagocytosis rates and (C) representative micrographs of peritoneal macrophages allowed to interact with GFP-expressing S. aureus (MOI 5), analyzed by imaging flow cytometry. Statistical evaluations were performed using the Mann–Whitney U test. Data were presented as mean± SEM or scatter dot plot with median. * = p<0.05; ns = not significant.

Fig 6. IL-1Ra treatment enhanced the mortality of staphylococcal sepsis.

NMRI mice inoculated with a sepsis dose of S. aureus strain Newman (2.2x10⁷ cfu/mouse) were treated with anakinra (IL-1Ra, 4mg/mouse, n = 10), or PBS (n = 10) every day starting on day 7 before inoculation with bacteria and continuing until the animals were sacrificed on day 14. Statistical evaluations were performed using the log-rank (Mantel-cox) test.