Deletion of Myeloid Interferon Regulatory Factor 4 (Irf4) in Mouse Model Protects against Kidney Fibrosis after Ischemic Injury by Decreased Macrophage Recruitment and Activation

AUTHORS

Kensuke Sasaki, Andrew S. Terker, Yu Pan, Zhilian Li, Shirong Cao, Yinqiu Wang, Aolei Niu, Suwan Wang, Xiaofeng Fan, Ming-Zhi Zhang and Raymond C. Harris

ABSTRACT

Background AKI is characterized by abrupt and reversible kidney dysfunction, and incomplete recovery leads to chronic kidney injury. Previous studies by us and others have indicated that macrophage infiltration and polarization play key roles in recovery from AKI. The role in AKI recovery played by IFN regulatory factor 4 (IRF4), a mediator of polarization of macrophages to the M2 phenotype, is unclear.

Methods We used mice with myeloid or macrophage cell–specific deletion of Irf4 (MΦ Irf4 −/−) to evaluate Irf4’s role in renal macrophage polarization and development of fibrosis after severe AKI.

Results Surprisingly, although macrophage Irf4 deletion had a minimal effect on early renal functional recovery from AKI, it resulted in decreased renal fibrosis 4 weeks after severe AKI, in association with less-activated macrophages. Macrophage Irf4 deletion also protected against renal fibrosis in unilateral ureteral obstruction. Bone marrow–derived monocytes (BMDMs) from MΦ Irf4 −/− mice had diminished chemotactic responses to macrophage chemoattractants, with decreased activation of AKT and PI3 kinase and increased PTEN expression. PI3K and AKT inhibitors markedly decreased chemotaxis in wild-type BMDMs, and in a cultured macrophage cell line. There was significant inhibition of homing of labeled Irf4 −/− BMDMs to postischemic kidneys. Renal macrophage infiltration in response to AKI was markedly decreased in MΦ Irf4 −/− mice or in wild-type mice with inhibition of AKT activity.

Conclusions Deletion of Irf4 from myeloid cells protected against development of tubulointerstitial fibrosis after severe ischemic renal injury in mice, due primarily to inhibition of AKT-mediated monocyte recruitment to the injured kidney and reduced activation and subsequent polarization into a profibrotic M2 phenotype.

Key indicators of repetitive overuse-induced neuromuscular inflammation and fibrosis are prevented by manual therapy in a rat model

AUTHORS

Mary F. Barbe, Michele Y. Harris, Geneva E. Cruz, Mamta Amin, Nathan M. Billett, Jocelynne T. Dorotan, Emily P. Day, Seung Y. Kim & Geoffrey M. Bove

ABSTRACT

Background

We examined the effectiveness of a manual therapy consisting of forearm skin rolling, muscle mobilization, and upper extremity traction as a preventive treatment for rats performing an intensive lever-pulling task. We hypothesized that this treatment would reduce task-induced neuromuscular and tendon inflammation, fibrosis, and sensorimotor declines.

Methods

Sprague-Dawley rats performed a reaching and lever pulling task for a food reward, 2 h/day, 3 days/week, for 12 weeks, while simultaneously receiving the manual therapy treatment 3 times per week for 12 weeks to either the task-involved upper extremities (TASK-Tx), or the lower extremities as an active control group (TASK-Ac). Results were compared to similarly treated control rats (C-Tx and C-Ac).

Results

Median nerves and forearm flexor muscles and tendons of TASK-Ac rats showed higher numbers of inflammatory CD68+ and fibrogenic CD206+ macrophages, particularly in epineurium, endomysium and epitendons than TASK-Tx rats. CD68+ and CD206+ macrophages numbers in TASK-Tx rats were comparable to the non-task control groups. TASK-Ac rats had more extraneural fibrosis in median nerves, pro-collagen type I levels and immunoexpression in flexor digitorum muscles, and fibrogenic changes in flexor digitorum epitendons, than TASK-Tx rats (which showed comparable responses as control groups). TASK-Ac rats showed cold temperature, lower reflexive grip strength, and task avoidance, responses not seen in TASK-Tx rats (which showed comparable responses as the control groups).

Conclusions

Manual therapy of forelimbs involved in performing the reaching and grasping task prevented the development of inflammatory and fibrogenic changes in forearm nerves, muscle, and tendons, and sensorimotor declines.

Bi-directional regulation functions of lanthanum-substituted layered double hydroxide nanohybrid scaffolds via activating...

AUTHORS

Min Chu, Zhenyu Sun, Zhanghao Fan, Degang Yu, Yuanqing Mao, Yaping Guo

ABSTRACT

Rationale: Osteoporotic patients suffer symptoms of excessive osteoclastogenesis and impaired osteogenesis, resulting in a great challenge to treat osteoporosis-related bone defects. Based on the positive effect of rare earth elements on bone metabolism and bone regeneration, we try to prove the hypothesis that the La3+ dopants in lanthanum-substituted MgAl layered double hydroxide (La-LDH) nanohybrid scaffolds simultaneously activate osteogenesis and inhibit osteoclastogenesis.

Methods: A freeze-drying technology was employed to construct La-LDH nanohybrid scaffolds. The in vitro osteogenic and anti-osteoclastogenic activities of La-LDH nanohybrid scaffolds were evaluated by using ovariectomized rat bone marrow stromal cells (rBMSCs-OVX) and bone marrow-derived macrophages (BMMs) as cell models. The in vivo bone regeneration ability of the scaffolds was investigated by using critical-size calvarial bone defect model of OVX rats.

Results: La-LDH nanohybrid scaffolds exhibited three-dimensional macroporous structure, and La-LDH nanoplates arranged perpendicularly on chitosan organic matrix. The La3+ dopants in the scaffolds promote proliferation and osteogenic differentiation of rBMSCs-OVX by activating Wnt/β-catenin pathway, leading to high expression of ALP, Runx-2, COL-1 and OCN genes. Moreover, La-LDH scaffolds significantly suppressed RANKL-induced osteoclastogenesis by inhibiting NF-κB signaling pathway. As compared with the scaffolds without La3+ dopants, La-LDH scaffolds provided more favourable microenvironment to induce new bone in-growth along macroporous channels.

Conclusion: La-LDH nanohybrid scaffolds possessed the bi-directional regulation functions on osteogenesis and osteoclastogenesis for osteoporotic bone regeneration. The modification of La3+ dopants in bone scaffolds provides a novel strategy for osteoporosis-related bone defect healing.

Gene expression of intracortical bone demonstrates loading-induced increases in Wnt1 and Ngf and inhibition of bone remodeling processes

AUTHORS

Taylor L.Harris, Matthew J.Silva

ABSTRACT

Osteocytes are the primary mechanosensitive cells in bone. However, their location in mineralized matrix has limited the in vivo study of osteocytic genes induced by mechanical loading. Laser Capture Microdissection (LCM) allows isolation of intracortical bone (Intra-CB), enriched for osteocytes, from bone tissue for gene expression analysis. We used microarray to analyze gene expression from mouse tibial Intra-CB dissected using LCM 4 h after a single loading bout or after 5 days of loading. Osteocyte enrichment was supported by greater expression of Sost, Dmp1, Dkk1, and Mepe in Intra-CB regions vs. Mixed regions containing periosteum and muscle (fold-change (FC) = 3.4, 2.2, 5.1, 3.0, respectively). Over 150 differentially expressed genes (DEGs) due to loading (loaded vs. contralateral control) in Intra-CB were found on Day 1 and Day 5, but only 10 genes were differentially expressed on both days, including Ngf (Day 1 FC = 13.5, Day 5 FC = 11.1) and Wnt1 (Day 1 FC = 1.5, Day 5 FC = 5.1). The expression of Ngf and Wnt1 within Intra-CB was confirmed by in situ hybridization, and a significant increase in number of Wnt1 mRNA molecules occurred on day 1. We also found changes in extracellular matrix remodeling with Timp1 (FC = 3.1) increased on day 1 and MMP13 (FC = 0.3) decreased on day 5. Supporting this result, IHC for osteocytic MMP13 demonstrated a marginal decrease due to loading on day 5. Gene Ontology (GO) biological processes for loading DEGs indicated regulation of vasculature, neuronal and immune processes while cell-type specific gene lists suggested regulation of osteoclast, osteoblast, and endothelial related genes. In summary, microarray analysis of microdissected Intra-CB revealed differential regulation of Ngf, Wnt1, and MMP13 due to loading in osteocytes.

Adenine-induced chronic kidney disease induces a similar skeletal phenotype in male and female C57BL/6 mice with more severe deficits in cortical bone properties of male mice

AUTHORS

Corinne E. Metzger, Elizabeth A. Swallow, Alexander J. Stacy, Matthew R. Allen

ABSTRACT

Chronic kidney disease (CKD) causes bone loss, particularly in cortical bone, through formation of cortical pores which lead to skeletal fragility. Animal models of CKD have shown variability in the skeletal response to CKD between males and females suggesting sex may play a role in this variation. Our aim was to compare the impact of adenine-induced CKD on cortical parameters in skeletally mature male and female C57Bl/6 mice. After 10-weeks of adenine-induced CKD, both male and female adenine mice had high serum parathyroid hormone (PTH), high bone turnover, and cortical porosity compared to non-CKD controls. Both sexes had lower cortical thickness, but only male mice had lower cortical bone area. CKD imparted greater deficits in mechanical properties of male mice compared to female mice. These data demonstrate that both male and female mice develop high PTH/high bone turnover in response to adenine-induced CKD and that cortical bone phenotypes are slightly more severe in males, particularly in mechanical properties deficits.

Cigarette smoke-associated inflammation impairs bone remodeling through NFκB activation

AUTHORS

Yi Lu, Yuanpu Peter Di, Ming Chang, Xin Huang, Qiuyan Chen, Ni Hong, Beth A. Kahkonen, Marissa E. Di, Chunyan Yu, Evan T. Keller & Jian Zhang

ABSTRACT

Background

Cigarette smoking constitutes a major lifestyle risk factor for osteoporosis and hip fracture. It is reported to impair the outcome of many clinical procedures, such as wound infection treatment and fracture healing. Importantly, although several studies have already demonstrated the negative correlation between cigarette consume and impaired bone homeostasis, there is still a poor understanding of how does smoking affect bone health, due to the lack of an adequately designed animal model. Our goal was to determine that cigarette smoke exposure impairs the dynamic bone remodeling process through induction of bone resorption and inhibition of bone formation.

Methods

We developed cigarette smoke exposure protocols exposing mice to environmental smoking for 10 days or 3 months to determine acute and chronic smoke exposure effects. We used these models, to demonstrate the effect of smoking exposure on the cellular and molecular changes of bone remodeling and correlate these early alterations with subsequent bone structure changes measured by microCT and pQCT. We examined the bone phenotype alterations in vivo and ex vivo in the acute and chronic smoke exposure mice by measuring bone mineral density and bone histomorphometry. Further, we measured osteoclast and osteoblast differentiation gene expression levels in each group. The function changes of osteoclast or osteoblast were evaluated.

Results

Smoke exposure caused a significant imbalance between bone resorption and bone formation. A 10-day exposure to cigarette smoke sufficiently and effectively induced osteoclast activity, leading to the inhibition of osteoblast differentiation, although it did not immediately alter bone structure as demonstrated in mice exposed to smoke for 3 months. Cigarette smoke exposure also induced DNA-binding activity of nuclear factor kappaB (NFκB) in osteoclasts, which subsequently gave rise to changes in bone remodeling-related gene expression.

Conclusions

Our findings suggest that smoke exposure induces RANKL activation-mediated by NFκB, which could be a “smoke sensor” for bone remodeling.