In Mouse Model, a TLR-9 Deficiency May Trigger Severe Lupus

Humoral Immune Changes

Given the increases and switch in specificity in the antibodies of Sle1TLR9^-/- mice, the authors went on to assess the humoral immune changes. They assessed B cell responses to TLR-7 in young mice that had not developed the disease.

Flow cytometry assays measuring B cell survival, activation and proliferation detected no differences between the two strains of mice. Further, they did not find increases in TLR-7 protein in young mouse B cells. However, samples collected on Day 4 revealed significantly higher amounts of IgG when stimulated.

Staining of freshly isolated splenocytes revealed Sle1TLR-9^-/- mouse B cells expressed more surface IgG than the Sle1 mice. The frequency of CD138+ plasma/plasmablast cells were increased with significantly higher TLR-7 levels when TLR-9 was absent.

They detected increased frequencies of splenic CD11b+ DCs with higher TLR-7 expression in experimental mice. This suggests TLR-7-high DCs have a role in the increase of CD138+ plasma/plasmablasts and IgG-switched B cells in negative mice. Because extrafollicular plasmablast and antibody switching in lupus have been attributed to DCs, this could be an interesting area to pursue.

Unexpected Results

Dr. Buyon

In SLE, the disease develops through chronic inflammation. Stimulation of TLR-7/TLR-9 results in immune activation through a common pathway. However, deletion in mice gives opposite results.

“Unexpectedly, TLR-9 deletion causes disease exacerbation in multiple spontaneous and induced lupus models,” says Dr. Fairhurst. “It is not known why. We found deletion of TLR-9 in our lupus-prone model resulted in up-regulation of TLR-7 expression and chronic disease.”

In particular, their data showed the pathogenesis of kidney disease was characterized by infiltrating renal conventional (c) DCs overexpressing TLR-7. The majority of infiltrating cells in the Sle1TLR-9^-/- mouse kidneys were myeloid (CD11b+). These were made up of MHCII-, F4/80+ macrophages and F4/80- cDCs. The MHCII- and the F4/80- cDCs increased in deficient mice compared with controls.

Kidney DCs or macrophages were pulsed with an antigen (ovalbumin; OVA) and then cultured with OVA-specific T cells to trigger activation. Renal cDCs from Sle1TLR9^-/- stimulated more T cell proliferation than cDCs from Sle1 mice. Renal macrophages showed no augmentation in T cell proliferation. Both renal DCs and macrophages had higher TLR-7 expression in the absence of TLR-9.

The researchers analyzed results from younger mice with less disease. Expression of TLR-7 was increased in the deficient mouse kidney F4/80+ macrophages and F4/80-^/low DCs. No increase was seen in MHCII- cells, similar to older mice.