Effects of a CCR2 antagonist on macrophages and Toll-like receptor 9 expression in a mouse model of diabetic nephropathy

The pathogenesis of diabetic nephropathy (DN) is related to macrophage (Mφ) recruitment to the kidneys, tumor necrosis factor-α (TNF-α) production, and oxidative stress. Toll-like receptor 9 (TLR9) activation is reportedly involved in systemic inflammation, and it exacerbates this condition in metabolic syndrome. Therefore, we hypothesized that TLR9 plays a role in the pathogenesis of DN. Two subsets of kidney macrophages in DN model (db/db) mice were analyzed using flow cytometry to evaluate their distribution and TLR9 expression and function. Mice were administered the CCR2 antagonist INCB3344 for 8 weeks; changes in macrophage distribution and function and its therapeutic effects on DN pathology were examined. Bone marrow-derived CD11bhigh (BM-) and tissue-resident CD11blow (Res-) Mφs were identified in the mouse kidneys. As DN progressed, the BM-Mφ number, TLR9 expression, and TNF-α production increased significantly. In Res-Mφs, reactive oxygen species (ROS) production and phagocytic activity were enhanced. INCB3344 decreased albuminuria, serum creatinine level, BM-Mφs abundance, TLR9 expression, and TNF-α production by BM-Mφs and ROS production by Res-Mφs. Both increased activation of BM-Mφs via TLR9 and TNF-α production and increased ROS production by Res-Mφs were involved in DN progression. Thus, inactivating macrophages and their TLR9 expressions by INCB3344 is a potential therapeutic strategy for diabetic nephropathy.

Therapeutic and Preventive Effects of Morphine Against Leishmania Major and Evaluation the Expression of TLRs and Cytokines in Infected Macrophages in Vitro and in BALB/c Mice

Purpose: Toll-Like Receptors (TLRs) and cytokines play key roles in infection control. They also enhance phagocytosis and killing of parasites. Morphine can modify host immunity and defense against infectious diseases. Methods: In this study, we assessed Therapeutic and preventive effects of morphine against Leishmania major and then we evaluated the expression of TLRs and pro and anti-inflamatory cytokines in both healthy macrophages and those infected with Leishmania major in vitro and in BALB/c mice. Results: Morphine showed preventive effect and no lesions were observed in the group that was taken morphine before being challenged with promastigotes. TLR2 expression decreased in drug-treated healthy macrophages, whereas TLR4 expression increased. TLR7 expression decreased in healthy macrophages. TLR9 expression was the highest in the groups treated with morphine in infected macrophages. Our findings revealed that healthy macrophages produce higher TNFα and lower IL6; the infected macrophages show a reverse pattern by producing higher IL6 and lower TNFα. We found that treatment with morphine strengthen defensive reactions against leishmaniasis. In mouse macrophages, the highest level of TLR9 expression was induced by morphine. Conclusion: TLR9 has critical role for recognition and control of microbial infection. No lesions were developed in mice treated with morphine before challenge which suggests a protective role for morphine in leishmaniasis. The positive role of morphine in decreasing IL-10 expression and increasing the expression of inflammatory cytokines such as TNF-α and therefore its preventing role in Leishmania disease.

TLR9 expression in Chronic Lymphocytic Leukemia identifies a pro-migratory subpopulation and novel therapeutic target.

CLL remains incurable despite BCR-targeted inhibitors revolutionizing treatment. This suggests that other signaling molecules are involved in disease escape mechanisms and resistance. Toll-like receptor 9 (TLR9) is a promising candidate, which is activated by unmethylated CpG-DNA. Here, we show that plasma from CLL patients contains significantly more unmethylated DNA than plasma from healthy controls (p<0.0001) and that cell-free DNA levels correlate with the prognostic markers CD38, b2-microglobulin and lymphocyte doubling time. Furthermore, elevated cell-free DNA was associated with shorter time to first treatment (TTFT: p=0.003, HR=4.0). We went on to show that TLR9 expression was associated with in-vitro CLL cell migration (p<0.001) and intracellular endosomal TLR9 strongly correlated with aberrant surface expression ((sTLR9); r=0.9). In addition, lymph node-derived CLL cells showed increased sTLR9 (p=0.016) and RNA sequencing of paired sTLR9hi and sTLR9lo CLL cells revealed differential transcription of genes involved in TLR signaling, adhesion, motility and inflammation in sTLR9hi cells. Mechanistically, the TLR9 agonist, ODN2006, promoted CLL cell migration (p<0.001) that was mediated, by p65 NF-kB and STAT3 transcription factor activation. Importantly, autologous plasma induced the same effects, which were reversed by a TLR9 antagonist. Furthermore, high TLR9 expression promoted engraftment and rapid disease progression in a NSG mouse xenograft model. Finally, we showed that dual targeting of TLR9 and BTK was strongly synergistic (median CI=0.2 at ED50), which highlights the distinct role for TLR9 signaling in CLL and the potential for combined targeting of TLR9 and BTK as a more effective treatment strategy in this incurable disease.

Study on H3K9 acetylation modification and TLR9 immune regulation mechanism in patients with anti-HBV treatment using thymosin a1 combined with entecavir

For hepatitis B antiviral treatment, there has been no comprehensive method yet. Interferon has poor antiviral efficacy, while nucleoside drugs have long course of treatment and high relapse rate. To improve the anti-HBV curative effect, treatment methods such as thymosin combined with entecavir have become a focus of clinical investigation. To explore potential mechanism of the combination therapy, based on previous studies, this paper explores the relationship between TLR9 expression in PBMCs, secretion of corresponding downstream inflammatory factors and HBV load in anti-HBV treatment with Thymosin a1 (Ta1) combined with entecavir. Chromatin immunoprecipitation combined with PCR method was adopted to detect H3K9 acetylation modification in patients. The relationship between TLR9 expression was explored using RT-QPCR, the relationship between secretion of inflammatory factors, efficacy and TLR9 mRNA expression was determined using Luminex technology. The results showed that during anti-HBV treatment with Ta1 combined with entecavir, histone acetylation increased in patients’ PBMCs, acetylated protein H3K9Ac had significant binding with promoter region of the TRL9 gene, thereby increasing the expression of TRL9 mRNA, activating the immune pathway under TRL9 regulation, promoting secretion of inflammation factors IL-6, IL-12, IFN-γ, and TNF-α, boosting the progress of antiviral therapy. H3K9 acetylation modification of TLR9 exists and plays an important role in patients with chronic hepatitis B. During the combination therapy with entecavir and Ta1, histone acetylation modification of TLR9 was significantly improved, which increased the expression of TLR9 at the mRNA and protein levels, and further regulated IL-6, IL-12 and other cytokines.