Figure 1.
Human beta-defensin 3 is up-regulated in the skin of leprosy patients with Type 1 reactions.
Skin biopsies from leprosy patients with Type 1 reactions (T1Rs) and leprosy patients without T1Rs (Controls) were assessed for their expression hBD2 and hBD3 as measured by qPCR. The skin of patients with T1Rs showed non-significantly elevated (A) hBD2 expression and a significant increase in (B) hBD3 expression. (C) Cellular infiltration in the T1Rs and the Controls show no significant differences. For the statistical analyses, values were first converted to a Gaussian distribution by taking their square and subsequently an un-paired t-test was performed. *p<0.05 is considered significant.
Figure 2.
Mycobacterium leprae induces human beta-defensins 2 and 3 in keratinocytes but not macrophages.
To determine whether keratinocytes would induce an antimicrobial program to M. leprae, immortalized keratinocytes (HaCaT cells) were stimulated with M. leprae whole cell sonicate at 1 µg/ml, 10 µg/ml, 100 µg/ml for 24 hours. M. leprae induced a dose-dependent up-regulation of hBD2 (A) and hBD3 (B) stable transcript levels as measured by qPCR. Macrophages cultured with M. leprae whole cell sonicate at 1 µg/ml, 10 µg/ml, 100 µg/ml for 24 hours showed no significant induction if hBD2 (C) and hBD3 (D). Experiments were performed at least twice in triplicate. **p<0.01, *p<0.05, n.s. (no significance) were determined by an unpaired t-test when compared to the controls.
Figure 3.
Mycobacterium leprae induces TLR2 but not TLR1 in keratinocytes.
Since activation of the TLR2/1 complex leads to keratinocytes expression of hBD2 and hBD3, increase in TLR2 and/or TLR1 may similarly lead to increased hBD expression. To evaluate whether the expression of TLR2 and TLR1 are induced by M. leprae, HaCaT cells were stimulated with 1 nM of the TLR2/1 agonist Pam3CSK4 or 100 µg/ml M. leprae for 24 hours. Both Pam3CSK4 and M. leprae significantly up-regulated stable transcript levels of (A) TLR2 but not (B) TLR1 in HaCaT cells. *p<0.05 or n.s. (no significance) were determined by an unpaired t-test when compared to the controls.
Figure 4.
Corticosteroids suppress human beta-defensins 2 and 3 in leprosy patients with Type 1 reactions.
The expression of hBD2 and hBD3 were assessed in skin biopsies of leprosy patients with Type 1 reactions before (day 0) and after (day 113) corticosteroid treatment. For hBD2, 13 out of 23 patients demonstrated suppression, with a mean transcript suppression of 78.5% when comparing day 113 to day 0 (A). For hBD3, 16 out of 23 patients demonstrated suppression, with a mean transcript suppression of 61.3% when comparing day 113 to day 0 (B).
Figure 5.
Prednisolone suppresses keratinocyte up-regulation of human beta-defensins 2 and 3 by M. leprae in vitro.
To determine whether prednisone suppresses keratinocyte expression of hBD2 and hBD3, HaCaT cells were stimulated with 100 µg/ml M. leprae and 0 (Control), 1, 10, and 100 nM of prednisone for 24 hours. Prednisolone caused a dose-dependent suppression hBD2 (A) and hBD3 (B). To confirm the suppression of hBD3 by prednisolone on the protein level, HaCaT cells were cultured in chamber slides with media alone (Control), 1 nM Pam3CSK4, 100 µg/ml M. leprae, or 100 µg/ml M. leprae and 100 nM prednisolone. Strong intracellular hBD3 expression is observed in cells treated with Pam3CSK4 or M. leprae. Prednisolone reduces the level of intracellular hBD3 produced by HaCaTs in response to M. leprae (C). IgG controls are represented by the bottom panel images. Significance (*p<0.05) was determined by an unpaired t-test.