Background/Purpose:

We previously demonstrated that repeated administration of the low molecular weight Toll-like receptor (TLR) 7 agonist (1V136) substantially reduces arthritic inflammation in mice.  Here we investigated the mechanisms contributing to this potent anti-inflammatory effect using the K/BxN passive transfer model of murine arthritis.

Methods: The following mutant strains were given arthritis with 150ul K/BxN serum intraperitoneally (ip) and treated with daily subcutaneous (s.c.) injections of vehicle or 150nmol 1V136 : C57BL/6, Tlr7^-/-, Ifnar1^-/-, kit^W-sh (Wsh), Pretty2, and Stat1. Bone marrow chimeric mice were generated by irradiating C57BL/6 and Tlr7^-/- mice with 10Gy and injecting these recipients with 10⁷ donor bone marrow cells (BM).  To assess the effects on vascular permeability mice were injected with 150ul K/BxN serum i.p. and 1mg/kg Evans blue dye intraveniously. After sacrifice the paws were removed and incubated in formamide overnight and the absorbance of the supernatant at 600nm was measured. Complete blood counts were performed by the UCSD Animal Care Program Diagnostic Laboratory. 

Results:

The joint swelling and arthritis scores of K/BxN serum transferred arthritis was significantly reduced by daily injections of 150nm 1V136 s.c. in C57BL/6 (WT) mice (AUC 6.6 drug treated vs. AUC 0.38 vehicle treated, P<0.001). Radiation bone marrow chimeric mice were tested and in WT BM>WT mice paw swelling in 1V136 treated mice was significantly less than vehicle treated controls (AUC 3.3 vs 7.4, P<0.01), but not in the TLR7 BM>TLR7 mice (AUC 6.7 vs 7.1, P>0.05). In mixed chimeras the drug was effective in the WT BM>TLR7 mice (AUC 4.9 vs 7.4, P=0.03), but not in the TLR7 BM>WT (AUC 6.2 vs 7.0, P>0.05). These data suggested that TLR7 is necessary for the activity of the compound. Mice that were defective in STAT1 and type I IFN signaling were refractory to 1V136 treatment, implicating these molecules effector molecules in the mechanism of this drug treatment. The data using BM chimeric mice suggested that radiosensitive cells were primarily involved in the beneficial effects of 1V136. A single dose of 1V136 reduced plasma extravasation in the joints of mice that received K/BxN sera.  Plasma extravasation in immune complex reactions is associated with mast cell degranulation and neutrophil recruitment. Two strains of mast cell deficient mice were tested: Pretty2 and kit^W-sh mice. The kit^W-sh mice were refractory to treatment (AUC 7.3 vs 5.6, P>0.05), but the Pretty2 mice responded to 1V136 (AUC 2.2 vs 7.5, P<0.05). In the Pretty 2 strain the c-kit mutation is also associated with a low level of circulating neutrophils. A single dose of 1V136 significantly decreased the circulating white blood cells in WT mice (10.6+0.22 vs 3.9+0.44, P<0.01), but not IFNAR1 null mice (11.77+0.7 vs 10.8+0.5, P<0.05). There was no effect in the platlet counts.

Conclusion:

The joint inflammation and vascular permeability observed in the passive serum transferred arthritis were significantly attenuated by 1V136 treatment.  The drug requires TLR7 on bone marrow derived cells, and an intact type I interferon pathway to be fully effective.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/treatment-of-innate-immune-arthritis-with-a-toll-like-receptor-7-agonist-requires-type-i-interferon/