Regional infusion of a class C TLR9 agonist enhances liver tumor microenvironment reprogramming and MDSC reduction to improve responsiveness to systemic checkpoint inhibition.

Myeloid-derived suppressor cells (MDSCs) expand in response to malignancy and suppress responsiveness to immunotherapy, including checkpoint inhibitors (CPIs). Within the liver, MDSCs have unique immunosuppressive features. While TLR9 agonists have shown promising activities in enhancing CPI responsiveness in superficial tumors amenable to direct needle injection, clinical success for liver tumors with TLR9 agonists has been limited by delivery challenges. Here, we report that regional intravascular infusion of ODN2395 into mice with liver metastasis (LM) partially eliminated liver MDSCs and reprogrammed residual MDSC. TLR9 agonist regional infusion also induced an increase in the M1/M2 macrophage ratio. Enhanced TLR9 signaling was demonstrated by an increased activation of in NFκB (pP65) and production of IL6 compared with systemic infusion. Further, PBMC-derived human MDSCs express TLR9, and treatment with class C TLR9 agonists (ODN2395 and SD101) reduced the expansion of MDSC population. TLR9 stimulation induced MDSC apoptosis and increased the M1/M2 macrophage ratio. Regional TLR9 agonist infusion along with systemic anti-PD-1 therapy improved control of LM. With effective delivery, TLR9 agonists have the potential to favorably reprogram the liver TME through reduction of MDSCs and favorable macrophage polarization, which may improve responsiveness to systemic CPI therapy.

Haematopoietic stem cells participate in muscle regeneration.

It has previously been shown that bone marrow cells contribute to skeletal muscle regeneration, but the nature of marrow cell(s) involved in this process is unknown. We used an immunocompetent and an immunocompromised model of bone marrow transplantation to characterize the type of marrow cells participating regenerating skeletal muscle fibres. Animals were transplanted with different populations of marrow cells from Green Fluorescent Protein (GFP) transgenic mice and the presence of GFP(+) muscle fibres were evaluated in the cardiotoxin-injured tibialis anterior muscles. GFP(+) muscle fibres were found mostly in animals that received either CD45(-), lineage(-), c-Kit(+), Sca-1(+) or Flk-2(+) populations of marrow cells, suggesting that haematopoietic stem cells (HSC) rather than mesenchymal cells or more differentiated haematopoietic cells are responsible for the formation of GFP(+) muscle fibres. Mac-1 positive population of marrow cells was also associated with the emergence of GFP(+) skeletal muscle fibres. However, most of this activity was limited to either Mac-1(+) Sca(+) or Mac-1(+)c-Kit(+) cells with long-term haematopoietic repopulation capabilities, indicating a stem cell phenotype for these cells. Experiments in the immunocompromised transplant model showed that participation of HSC in the skeletal muscle fibre formation could occur without haematopoietic chimerism.

Regulation of lens aldose reductase activity by nitric oxide.

To examine the regulation of aldose reductase (AR) activity by nitric oxide (NO) in human lens epithelial cells (HLEC), cultured rat lens, and normal and diabetic rat lens, we have incubated HLEC or cultured rat lenses with 1 mm of the NO donors S-nitroso-N-acetylpenicillamine (SNAP) or S-nitrosoglutathione (GSNO), and the AR activity and sorbitol content were measured. Non-diabetic and diabetic (treated with streptozotocin 65 mg kg(-1) body wt, i.p.) rats were injected with the nitric oxide synthase (NOS) inhibitor, L-NAME (50 mg kg(-1) body wt day(-1), x 10 days i.p.) or NOS substrate, L-arginine (200 mg kg(-1) body wt day(-1), x 10 days i.p.). In a separate group of rats, a nitroglycerin (NG)-patch that releases 200 ng min(-1) NO was applied to the dorsal neck region. After 10 days of treatment, the lenses were removed and their AR activity and sorbitol content were measured. Incubation of HLEC with SNAP or GSNO reduced AR activity. A similar reduction in AR activity and sorbitol accumulation was observed when diabetic and non-diabetic rat lenses were cultured in the presence of SNAP and GSNO. Total protein-SSG in diabetic lens was lower compared to normal lens. Treatment of diabetic and non-diabetic rats with L-NAME enhanced AR activity and sorbitol accumulation, whereas NG patch and L-arginine significantly decreased AR activity and sorbitol accumulation in diabetic lenses compared to non-diabetic. Increased S-glutathiolation of AR was observed in the presence of SNAP. These results suggest that decreased glutathiolation of cellular proteins in diabetic rat lens compared to non-diabetic lens is related to decreased NO availability in diabetic rats which would decrease GSNO. Restoring the NO levels in diabetic animals increases glutathiolation of cellular proteins, inhibits AR activity and prevents sorbitol accumulation. Exogenous delivery of NO may represent a potentially useful strategy for preventing or delaying diabetic cataractogenesis and the development of other diabetic complications.

Critical variables in the conversion of marrow cells to skeletal muscle.

We have studied conversion of marrow cells to skeletal muscle in cardiotoxin-injured anterior tibialis muscle in a green fluorescent protein (GFP) to C57BL/6 transplantation model and ascertained that total body irradiation (TBI) with establishment of chimerism is a critical factor. Local irradiation has little effect in lower doses and was detrimental at higher doses. Whole body (1000 cGy) with shielding of the leg or a combination of 500 cGy TBI and 500 cGy local radiations was found to give the best results. In non-obese diabetic-severe combined immunodeficient (NOD-SCID) recipients, we were able to show that conversion could occur without radiation, albeit at relatively lower levels. Within 3 days of cardiotoxin injury, GFP-positive mononuclear cells were seen in the muscle, and within 2 weeks GFP-positive muscle fibers were identified. Conversion rates were increased by increasing donor-cell dose. Timing of the cardiotoxin injury relative to the transplantation was critical. These studies show that variables in transplantation and injury are critical features of marrow-to-muscle conversions. Irradiation primarily effects conversion by promoting chimerism. These data may explain the differences in the literature for the frequency of marrow-to-skeletal muscle conversion and can set a platform for future models and perhaps clinical protocols.

Robust conversion of marrow cells to skeletal muscle with formation of marrow-derived muscle cell colonies: a multifactorial process.

OBJECTIVE: Murine marrow cells are capable of repopulating skeletal muscle fibers. A point of concern has been the "robustness" of such conversions. We have investigated the impact of type of cell delivery, muscle injury, nature of delivered cell, and stem cell mobilizations on marrow-to-muscle conversion. METHODS: We transplanted green fluorescence protein (GFP)-transgenic marrow into irradiated C57BL/6 mice and then injured anterior tibialis muscle by cardiotoxin. One month after injury, sections were analyzed by standard and deconvolutional microscopy for expression of muscle and hematopoietic markers. RESULTS: Irradiation was essential to conversion, although whether by injury or induction of chimerism is not clear. Cardiotoxin- and, to a lesser extent, PBS-injected muscles showed significant number of GFP(+) muscle fibers, while uninjected muscles showed only rare GFP(+) cells. Marrow conversion to muscle was increased by two cycles of G-CSF mobilization and to a lesser extent by G-CSF and steel or GM-CSF. Transplantation of female GFP to male C57BL/6 and GFP to ROSA26 mice showed fusion of donor cells to recipient muscle. High numbers of donor-derived muscle colonies and up to 12% GFP(+) muscle cells were seen after mobilization or direct injection. These levels of donor muscle chimerism approach levels that could be clinically significant in developing strategies for the treatment of muscular dystrophies. CONCLUSION: In summary, the conversion of marrow to skeletal muscle cells is based on cell fusion and is critically dependent on injury. This conversion is also numerically significant and increases with mobilization.

hRad9 rapidly binds DNA containing double-strand breaks and is required for damage-dependent topoisomerase II beta binding protein 1 focus formation.

Checkpoint proteins protect the genomic integrity of a cell, repeatedly impaired by DNA damage and normal cellular processes, such as replication. Checkpoint proteins hRad9, hRad1, and hHus1 form a heterotrimeric complex that is thought to act as a genomic surveyor of DNA damage. We show here that, when DNA double-strand breaks (DSBs) are specifically generated in a subnuclear area, hRad9 is rapidly retained at the damaged DNA, within 2 min of damage induction. Rapid localization of hRad9 to regions of DNA containing DSBs is most efficient during replication. Furthermore, hRad9 colocalizes with the phosphorylated form of damage-response protein H2AX (gamma H2AX) after DNA damage. This localization is independent of the damage repair kinase ataxia telangiectasia-mutated kinase (ATM), because hRad9/gamma H2AX colocalization still occurs in ATM(-/-) fibroblasts. Secondly, hRad9 interacts with replication and checkpoint protein topoisomerase II beta binding protein 1 (TopBP1) before and after DNA damage, and this interaction is dependent on the COOH-terminal 17 amino acids of hRad9. Overexpression of a COOH-terminally deleted form of hRad9 abolishes the colocalization of TopBP1 to gamma H2AX, ablating TopBP1 but not gamma H2AX foci formation. The loss of TopBP1 containing foci, but not of gamma H2AX containing foci, indicates that hRad9 is required for TopBP1 focus formation after damage, but is not required for gamma H2AX formation at DSBs. These results are consistent with a model in which the hRad9/hHus1/hRad1 complex acts as a checkpoint sensor during S phase by rapidly localizing to sites of DNA damage and transducing checkpoint responses by facilitating proper localization of downstream checkpoint proteins, including TopBP1.