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Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells
Published on: August 5, 2016
Brucella melitensis clinical isolate modulates osteoclast differentiation to drive pathological bone destruction in
Jinlei Chen1,2,3,4, Feijie Zhi2,3,4, Guanghai Zhao1
1Orthopedics Key Laboratory of Gansu Province, Department of Orthopedics, The Cuiying Biomedical Research Center, The Second Hospital & Clinical Medical School, Lanzhou University, Lanzhou, China.
Background:
Brucellosis is a widespread zoonosis that is acquired by humans from infected animals. Articular complications, particularly brucellar spondylitis, are the most prevalent and disabling manifestations of human brucellosis. Inflammation-mediated osteoclast activation is implicated in Brucella-induced bone destruction, but the direct cellular tropism of Brucella within bone tissue and the specific effects of infection on osteoclasts remain poorly understood. This study aims to characterize the osteoclast tropism of Brucella melitensis biovar 3 clinical isolates and their direct regulatory effects on osteoclast-mediated bone destruction in Brucella-induced arthritis.
Methods:
Brucella clinical isolates were obtained from the bone tissues of human brucellar spondylitis patients in Gansu Province, China. Whole-genome sequencing and biotyping identified their specific biovars. These isolates were used to generate arthritis in immunodeficient NCG mice; bone homeostasis in these mice was assessed via ELISA. We assessed their cellular tropism and osteoclast-modulating effects through intracellular survival assays, immunofluorescence, histopathology, TRAP staining, and resorption pit analysis.
Results:
Three clinical isolates of B. melitensis biovar 3 were obtained from arthritis lesions in patients from Gansu. Genomic analysis revealed homology with geographically diverse Chinese Brucella strains. Although these isolates reached splenic bacterial loads similar to the virulent strain 16M, they did not cause splenomegaly by two weeks post-infection. The isolates displayed strong tropism for human and murine osteoclasts, achieving significantly higher intracellular loads compared to osteoblasts or osteocytes. Infection at the osteoclast precursor/bone marrow macrophage stage enhanced early osteoclastogenesis while inhibiting late-stage apoptosis and fusion, leading to prolonged osteoclast survival and aggravated bone resorption and defects. In contrast, conditioned medium from infected osteoblasts or osteocytes had minimal impact on late-stage osteoclast differentiation.
Conclusions:
These findings elucidate the mechanisms underlying pathological bone defects in brucellar arthritis. The direct bacterial effects, together with the formation of an osteoclast-derived pro-survival niche, account for the prevalence of brucellar arthritis as the most common complication of chronic brucellosis. Targeting the interaction between B. melitensis and osteoclasts may thus offer a novel therapeutic strategy for preventing and treating Brucella-induced osteolytic lesions.
Insights
Brucella melitensis directly infects and targets osteoclasts, leading to prolonged survival and bone destruction in brucellar arthritis. This interaction explains the high prevalence of this disabling complication of brucellosis.
Area of Science:
- Microbiology
- Immunology
- Bone Biology
Background:
- Brucellosis is a zoonotic disease with articular complications like brucellar spondylitis.
- Osteoclast activation is implicated in Brucella-induced bone destruction, but bacterial tropism and effects on osteoclasts are unclear.
Purpose of the Study:
- Characterize the osteoclast tropism of Brucella melitensis biovar 3 clinical isolates.
- Investigate direct regulatory effects on osteoclast-mediated bone destruction in brucellar arthritis.
Main Methods:
- Obtained Brucella clinical isolates from human spondylitis patients.
- Used whole-genome sequencing, biotyping, and mouse models.
- Assessed cellular tropism and osteoclast effects via various in vitro and in vivo assays.
Main Results:
- Identified B. melitensis biovar 3 isolates with genomic homology to Chinese strains.
- Demonstrated strong tropism for human and murine osteoclasts over osteoblasts/osteocytes.
- Showed infection enhances osteoclastogenesis, inhibits apoptosis, prolongs survival, and increases bone resorption.
Conclusions:
- Elucidated mechanisms of bone defects in brucellar arthritis.
- Highlighted osteoclast-derived pro-survival niche in chronic brucellosis.
- Proposed targeting B. melitensis-osteoclast interaction as a therapeutic strategy.
