Acne patients who were scheduled by their dermatologist to begin treatment with isotretinoin for severe acne were recruited. Normal healthy volunteers without acne or prior history of isotretinoin exposure served as controls. A total of 27 acne patients, 10 males and 17 females, with a mean (± SEM) age of 17.9 ± 1.4 years and 23 normal volunteers, 8 males and 15 females, with a mean (± SEM) age of 22.4 ± 1.5 years were enrolled. Of the 27 acne patients treated with isotretinoin, 10 completed the study through 20 weeks of isotretinoin therapy, eight of whom were reevaluated six months after completing therapy. Of the remaining patients, one discontinued isotretinoin at the direction of his dermatologist, two discontinued due to medication costs, one withdrew consent, and six were lost to follow-up between eight and 20 weeks.
Acne lesion counts were performed at all visits to assess treatment response (Figure S1
). As an indicator of compliance, serum levels of isotretinoin and its isomers ATRA and 9-cis
retinoic acid (9-cis
RA) were assessed (Figure S2
). Levels were also measured in normal volunteers for comparison. No differences in serum levels of isotretinoin, ATRA, or 9-cis
RA were found between acne patients at baseline and normal volunteers.
Acne patients’ monocytes express significantly greater levels of TLR-2 and significantly lower levels of TLR-4 compared to normal volunteers
Monocytes from acne patients secrete significantly higher levels of cytokines when exposed to P. acnes
compared to monocytes from normal subjects (Sugisaki et al., 2009
). We sought to test the hypothesis that monocytes from acne patients express higher levels of TLR-2 when exposed to P. acnes
, which may account for this observation. At baseline, acne patients’ unstimulated monocytes expressed higher levels of TLR-2 than normal volunteers (p = 0.036) (). Treatment with P. acnes
sonicate induced TLR-2 expression in both patients’ and volunteers’ monocytes, but expression was significantly greater in acne patients’ monocytes (p = 0.041). In contrast, P. acnes
-stimulated monocytes from acne patients at baseline expressed significantly lower levels of TLR-4 compared to normal volunteers (p = 0.016) ().
Isotretinoin therapy down-regulates surface expression of TLR-2 on patients’ monocytes in vivo
Acne patients’ monocytes express lower levels of TLR-4 compared to normal volunteers
Isotretinoin down-regulates cell surface expression of TLR-2, but not TLR-4, on patients’ monocytes in vivo
To assess the ability of systemic isotretinoin therapy to modulate TLR expression in vivo, peripheral blood samples were taken from acne patients prior to and during isotretinoin therapy for acne (). Compared to baseline, unstimulated monocytes from patients treated with isotretinoin had significantly decreased TLR-2 expression as early as 1 week of treatment (p < 0.001), which continued to decrease at each time-point examined (4 weeks, p = 0.004; 8 weeks, p = 0.001; 20 weeks, p = 0.040) (). Similarly, decreased TLR-2 expression was noted when monocytes from patients treated with isotretinoin were stimulated with P. acnes sonicate (1 week, p = 0.004; 4 weeks, p = 0.017; 8 weeks, p = 0.001; 20 weeks, p = 0.006) () or whole heat-killed P. acnes (data not shown). At the 6 month follow-up, the mean fluorescence intensity for TLR-2 expression in stimulated and unstimulated monocytes from the 8 subjects decreased by approximately 70% compared to baseline. In comparison, isotretinoin therapy did not affect TLR-4 expression on acne patients’ monocytes at any point during therapy ().
Since the majority of patients treated with a full course of isotretinoin (approximately 20 weeks) have a permanent remission of their acne (Liu et al., 2008a
), we sought to test the hypothesis that the dramatic reduction in monocyte TLR-2 expression persists following cessation of isotretinoin therapy. To do this, we modified our clinical protocol and were able to reexamine 8 of the 10 patients that completed 20 weeks of therapy. Six months after stopping isotretinoin, TLR-2 expression in unstimulated and P. acnes
-stimulated monocytes was still significantly decreased compared to baseline (p = 0.004 and 0.0004, respectively), and not significantly different from normal volunteers (). Additionally, TLR-4 expression in monocytes from acne patients six months after treatment was no longer significantly lower than normal volunteers.
Isotretinoin therapy decreases inflammatory cytokine production by patients’ monocytes in response to P. acnes
To determine if isotretinoin altered monocyte cytokine response to P. acnes stimulation, supernatants from cultured monocytes were assayed for inflammatory cytokines using bead arrays. Prior to isotretinoin therapy, acne patients’ unstimulated monocytes secreted more TNFα than monocytes from normal volunteers, but due to inter-patient variability, did not differ in secretion of other inflammatory cytokines (). In response to stimulation with P. acnes sonicate, acne patients’ monocytes at baseline secreted more IL-1β, IL-6, IL-10, and IL-12p70 than monocytes from normal volunteers. Isotretinoin therapy significantly decreased the secretion of IL-1β, IL-6, IL-10, and IL-12p70 by acne patients’ monocytes in response to P. acnes compared to pretreatment levels. Cytokine secretion decreased as early as one week of isotretinoin therapy and continued to decrease through 20 weeks of therapy. Additionally, this decrease was sustained at six months after the cessation of therapy. Pearson correlation analysis showed high correlations between TLR-2 expression and secretion of IL-1β (p < 0.000), IL-6 (p < 0.000), IL-8 (p = 0.001), and IL-10 (p < 0.000), but not IL-12p70 (p > 0.1) in P. acnes-stimulated monocytes from volunteers and acne patients during isotretinoin therapy. Similar results were observed in monocytes treated with whole heat-killed P. acnes (data not shown).
Isotretinoin therapy decreases inflammatory cytokine production by acne patients’ monocytes in response to P. acnes
Isotretinoin therapy does not alter the proportion or function of circulating Treg
Treg suppress inflammatory responses and are of great interest as potential therapies for autoimmune and chronic inflammatory diseases. Because the peripheral conversion of undifferentiated T cells into Treg may involve ATRA (Mucida et al., 2007
; Wang et al., 2009
; Xiao et al., 2008
), as part of the original study we also sought to test the hypothesis that isotretinoin might increase the proportion of circulating Treg in treated patients. Peripheral proportions of CD4+CD25+Foxp3+ Treg were not significantly different in acne patients during isotretinoin therapy compared to baseline (Figure S3
). Additionally, we found no differences between acne patients and normal volunteers in peripheral proportions of Treg. To investigate whether in vivo
priming with isotretinoin renders peripheral naïve T cells more likely to differentiate into Treg upon activation, lymphocytes were also stimulated for five days with anti-CD3 and anti-CD28 antibodies, P. acnes
sonicate, or vehicle. Acne patients’ T cells during isotretinoin therapy were not more likely to differentiate into Treg upon stimulation compared to baseline (data not shown). These data suggest that systemic isotretinoin therapy does not necessarily impart Treg-differentiating signals on naïve T cells in the periphery in vivo
To determine if isotretinoin affects peripheral Treg suppressive function, peripheral lymphocyte proliferation was measured using 3H-thymidine incorporation assays. Isotretinoin therapy did not affect proliferation of acne patients’ lymphocytes at either an unstimulated basal level or in response to activation by anti-CD3/anti-CD28 antibodies or P. acnes sonicate (). Interestingly, in response to P. acnes antigen, acne patients had significantly suppressed lymphocyte proliferation compared to normal volunteers (p= 0.009). Taken together, these data suggest that acne patients have a blunted lymphocyte proliferative response to P. acnes and that treatment with isotretinoin does not alter the proportion or function of circulating Treg in acne patients.
Lymphocyte proliferative response to P.acnes is blunted in acne patients and is unaffected by isotretinoin
Acne patients have higher serum concentrations of IL-10, but not inflammatory cytokines, compared to healthy volunteers
Aside from suppression by Treg, lymphocyte proliferation can also be inhibited by immunosuppressive cytokines such as IL-10. Serum was collected from all subjects during the study for assay of cytokine levels. At baseline, acne patients had significantly higher levels of serum IL-10 compared to normal volunteers (P = 0.002) (). Interestingly, isotretinoin did not affect serum IL-10 levels at any time-point, and serum IL-10 remained elevated throughout therapy. Of note, these levels are low and their clinical significance is unknown. In contrast, acne patients did not differ from normal volunteers in serum concentrations of IL-1β, IL-6, IL-8, or IL-12p70, nor did isotretinoin therapy significantly affect serum concentrations of these cytokines.
Acne patients have higher serum levels of IL-10 compared to normal subjects
Isotretinoin therapy does not affect cytokine secretion by peripheral blood lymphocytes
Retinoid treatment of T cells can skew helper T cell differentiation and responses. To investigate the effects of systemic isotretinoin therapy on the secretion of TH1, TH2, and TH17-associated cytokines by peripheral lymphocytes, media supernatants from P. acnes- and anti-CD3/anti-CD28-stimulated lymphocytes were analyzed at 20 hours and five days of treatment as part of the original study. Isotretinoin did not significantly affect IL-2, IL-4, IL-5, IL-10, IL-17A, TNFα, or IFNγ secretion by lymphocytes in any treatment group (data not shown), suggesting that T cell differentiation was unaffected. Additionally, we did not find any difference in proportions of Treg, Th2, or Th17 cells as assessed by flow cytometry of patients’ lymphocytes (data not shown).