Activation of Heme Metabolism Promotes Tissue Health After Intraarticular Injury or Surgical Exposure.

Posttraumatic osteoarthritis (PTOA) is a well-recognized public health burden without any disease modifying treatment. This occurs despite noted advances in surgical care in the past 50 years. Mitochondrial oxidative damage pathways initiate PTOA after severe injuries like intraarticular fracture that often require surgery and contribute to PTOA after less severe injuries that may or may not require surgery like meniscal injuries. When considering the mitochondrial and redox environment of the injured joint, we hypothesized that activation of heme metabolism, previously associated with healing in many settings, would cause prototypic mitochondrial reprogramming effects in cartilage ideally suited for use at the time of injury repair. Activation of heme metabolism can be accomplished through the gasotransmitter carbon monoxide (CO), which activates hemeoxygenase-1 (HO1) and subsequent heme metabolism. In this study, we employed unique carbon monoxide (CO)-containing foam (COF) to stimulate heme metabolism and restore chondrocyte oxygen metabolism in vitro and in vivo . Doxycycline-inducible, chondrocyte-specific HO1 overexpressing transgenic mice show similar mitochondrial reprogramming after induction compared to COF. CO is retained at least 24 h after COF injection into stifle joints and induces sustained increases in heme metabolism. Lastly, intraarticular injection of COF causes key redox outcomes without any adverse safety outcomes in rabbit stifle joints ex vivo and in vivo . We propose that activation of heme metabolism is an ideal adjuvant to trauma care that replenishes chondrocyte mitochondrial metabolism and restores redox homeostasis.

A reciprocal relationship between mitochondria and lipid peroxidation determines the chondrocyte intracellular redox environment.

In orthopedic research, many studies have applied vitamin E as a protective antioxidant or used tert-butyl hydroperoxide to induce oxidative injury to chondrocytes. These studies often support the hypothesis that joint pathology causes oxidative stress and increased lipid peroxidation that might be prevented with lipid antioxidants to improve cell survival or function and joint health; however, lipid antioxidant supplementation was ineffective against osteoarthritis in clinical trials and animal data have been equivocal. Moreover, increased circulating vitamin E is associated with increased rates of osteoarthritis. This disconnect between benchtop and clinical results led us to hypothesize that oxidative stress-driven paradigms of chondrocyte redox function do not capture the metabolic and physiologic effects of lipid antioxidants and prooxidants on articular chondrocytes. We used ex vivo and in vivo cartilage models to investigate the effect of lipid antioxidants on healthy, primary, articular chondrocytes and applied immuno-spin trapping techniques to provide a broad indicator of high levels of oxidative stress independent of specific reactive oxygen species. Key findings demonstrate lipid antioxidants were pro-mitochondrial while lipid prooxidants decreased mitochondrial measures. In the absence of injury, radical formation was increased by lipid antioxidants; however, in the presence of injury, radical formation was decreased. In unstressed conditions, this relationship between chondrocyte mitochondria and redox regulation was reproduced in vivo with overexpression of glutathione peroxidase 4. In mice aged 18 months or more, overexpression of glutathione peroxidase 4 significantly decreased the presence of pro-mitochondrial peroxisome proliferation activated receptor gamma and deranged the relationship between mitochondria and the redox environment. This complex interaction suggests strategies targeting articular cartilage may benefit from adopting more nuanced paradigms of articular chondrocyte redox metabolism.

The aged microenvironment impairs BCL6 and CD40L induction in CD4+ T follicular helper cell differentiation.

Weakened germinal center responses by the aged immune system result in diminished immunity against pathogens and reduced efficacy of vaccines. Prolonged contacts between activated B cells and CD4+ T cells are crucial to germinal center formation and T follicular helper cell (Tfh) differentiation, but it is unclear how aging impacts the quality of this interaction. Peptide immunization confirmed that aged mice have decreased expansion of antigen-specific germinal center B cells and reduced antibody titers. Furthermore, aging was associated with accumulated Tfh cells, even in naïve mice. Despite increased numbers, aged Tfh had reduced expression of master transcription factor BCL6 and increased expression of the ectonucleotidase CD39. In vitro activation revealed that proliferative capacity was maintained in aged CD4+ T cells, but not the costimulatory molecule CD40L. When activated in vitro by aged antigen-presenting cells, young CD4+ naïve T cells generated reduced numbers of activated cells with upregulated CD40L. To determine the contribution of cell-extrinsic influences on antigen-specific Tfh induction, young, antigen-specific B and CD4+ T cells were adoptively transferred into aged hosts prior to peptide immunization. Transferred cells had reduced expansion and differentiation into germinal center B cell and Tfh and reduced antigen-specific antibody titers when compared to young hosts. Young CD4+ T cells transferred aged hosts differentiated into Tfh cells with reduced PD-1 and BCL6 expression, and increased CD39 expression, though they maintained their mitochondrial capacity. These results highlight the role of the lymphoid microenvironment in modulating CD4+ T cell differentiation, which contributes to impaired establishment and maintenance of germinal centers.