Fig. 6.

Effect of genotype and treatment with HA or PEP-1 on Lgr5⁺ cell number and proliferation. A: Lgr5+ cell number. Effect of genotype was measured; 14-day-old WT and CD44^−/− mice had similar numbers of Lgr5⁺ crypt epithelial cells per section in nonfissioning and fissioning crypts. TLR4^−/− mice also had a similar number of Lgr5⁺ epithelial cells at baseline in nonfissioning crypts but had significantly fewer Lgr5⁺ epithelial cells in fissioning crypts compared with WT or CD44^−/− mice. Overall Lgr5⁺ cell numbers were significantly diminished in 14-day-old TLR4^−/− mice compared with WT and CD44^−/− mice, especially in fissioning crypts. +P < 0.01, ++P < 0.001, +++P < 0.0001 compared with WT mice given the same treatment. Effect of treatment was measured; postnatal WT and CD44^−/− mice showed a small increase in Lgr5⁺ cell numbers in nonfissioning crypts after treatment with exogenous HA from age 7 days to age 14 days, but there was no increase in TLR4^−/− mice. Blocking endogenous HA by intraperitoneal administration of PEP-1 from age 7 days to age 14 days had no effect on Lgr5⁺ cell numbers in WT or CD44^−/− mice, but there was a small further diminishment of Lgr5⁺ cell numbers in TLR4^−/− mice. Values are means ± SE for 6 mice per treatment group. *P < 0.01 compared with control of the same genotype. B: Lgr5⁺ intestinal crypt epithelial cell proliferation. Effect of genotype was measured, and 14-day-old WT and CD44^−/− mice had similar baseline rates of Lgr5⁺ intestinal crypt epithelial cell proliferation, with higher percentages of Lgr5⁺ epithelial cells proliferating in fissioning than in nonfissioning crypts. 14-day-old TLR4^−/− mice had significantly lower rates of Lgr5⁺ epithelial cell proliferation compared with WT and CD44^−/− mice. TLR4^−/− mice also differed from WT and CD44^−/− mice in having a rate of Lgr5⁺ epithelial proliferation that was no greater in fissioning crypts than in the nonfissioning crypts. +P < 0.01, ++P < 0.001, +++P < 0.0001 compared with WT mice given the same treatment. Effect of treatment was measured, and mice were treated with HA or PEP-1 as in Fig. 4. Treatment with HA had no effect on Lgr5⁺ epithelial cell proliferation in WT, CD44^−/−, or TLR4^−/− mice. Administration of PEP-1 resulted in a significant decrease in Lgr5⁺ cell proliferation in nonfissioning intestinal crypts in WT and CD44^−/− mice, while not affecting Lgr5⁺ cell proliferation in fissioning crypts. PEP-1 had no effect on Lgr5⁺ epithelial cell proliferation in TLR4^−/− mice. Values are means ± SE for 6 mice per treatment group. *P < 0.01 compared with control of the same genotype. C–J: immunofluorescence for BrdU (red) + Lgr5 (GFP, green) showing BrdU-labeled and Lgr5⁺ epithelial cells in nonfissioning and fissioning small intestinal crypts of postnatal day 14 WT (C–F) and TLR4^−/− (G–J) mice. Scale bar for C, E, G, I = 50 μm, original magnification ×400. Scale bar for D, F, H, J = 20 μm, original magnification ×1,000.