p38 MAP Kinase Inhibition Reduces Propionibacterium acnes

14 Pages·2015·1.85 MB·English

DermatolTher(Heidelb)(2015)5:53–66
DOI10.1007/s13555-015-0072-7
ORIGINAL RESEARCH
p38 MAP Kinase Inhibition Reduces Propionibacterium
acnes-Induced Inflammation in Vitro
Wen-HwaLi • LiZhang •Peter Lyte• Karien Rodriguez •
Druie Cavender• Michael D.Southall
Toviewenhancedcontentgotowww.dermtherapy-open.com
Received:January26,2015/Publishedonline:March7,2015
(cid:2)TheAuthor(s)2015.ThisarticleispublishedwithopenaccessatSpringerlink.com
ABSTRACT Phosphorylation of MAPKs without or with
p38 inhibitors was examined by Western blot
Introduction: Propionibacterium acnes, a andcytokinesecretionwasdetectedbyEnzyme-
ubiquitous skin bacterium, stimulates Linked Immunosorbent Assay (ELISA).
keratinocytes to produce a number of Results: Increased levels of phospho-p38 were
proinflammatory cytokines and may observedinhumanacnelesions,predominantly
contribute to inflammatory acne. The aim of in follicular and perifollicular keratinocytes.
the study was to investigate whether P. acnes- Exposure of cultured human keratinocytes to
induced proinflammatory cytokine release is viable P. acnes resulted in phosphorylation of
mediated by P. acnes-induced activation of p38 multiple members of the MAPK family,
mitogen-activatedproteinkinase (p38MAPK or including rapid and transient activation of p38
p38) in human keratinocytes. and extracellular signal-related kinase (ERK1/2)
Methods: Immunohistochemistry was used to and relatively slow but sustained activation of
evaluate p38 phosphorylation in human skin c-Jun N-terminal kinases (JNK1/2). Viable P.
samples with or without acne. Primary human acnes induced the secretion of interleukin-1a
keratinocytes and epidermal skin equivalents (IL-1a), tumor necrosis factor-a (TNF-a), and
were exposed to viable P. acnes. IL-8 from human keratinocytes. The
phosphorylation of p38 (phospho-p38) and
Electronicsupplementarymaterial Theonline the secretion of cytokines induced by P. acnes
versionofthisarticle(doi:10.1007/s13555-015-0072-7)
in cultured keratinocytes were inhibited by
containssupplementarymaterial,whichisavailableto
authorizedusers. SB203580, a p38a/b inhibitor. Furthermore,
SCIO-469, a selective inhibitor of p38a,
W.-H.Li(&)(cid:2)L.Zhang(cid:2)P.Lyte(cid:2)K.Rodriguez(cid:2)
showed similar effects in cultured
D.Cavender(cid:2)M.D.Southall
DepartmentofSkinBiologyandPharmacology,The keratinocytes. Topical treatment of SCIO-469
Johnson&JohnsonSkinResearchCenter,Johnson
inhibited the P. acnes-induced phospho-p38
&JohnsonConsumerandPersonalProducts
Worldwide,DivisionofJohnsonandJohnson and cytokine secretion in human epidermal
ConsumerCompanies,Inc.,199GrandviewRoad,
equivalents.
Skillman,NJ08558,USA
e-mail:[email protected]
54 DermatolTher(Heidelb)(2015)5:53–66
Conclusion: ThedatademonstratethatP.acnes by proinflammatory cytokines and stress-
inducesp38-dependentinflammatoryresponses inducing stimuli [33]. For p38 MAPKs, a and b
inkeratinocytes,andsuggestthatp38mayplay are ubiquitously expressed, whereas c and d are
an important role in the pathogenesis of expressed in more restricted patterns [37]. In
inflammatory acne. human keratinocytes, p38a and b are involved
Funding: Johnson & Johnson. in responses to stress and proinflammatory
cytokines, while p38d has been implicated in
Keywords: Acne vulgaris; Inflammation; keratinocyte differentiation [38]. p38 MAPKs
Keratinocyte; p38 mitogen-activated protein have been implicated in inflammatory disease
kinase; p38 inhibitor; Propionibacterium acnes pathways such as psoriasis, asthma, and
Crohn’s disease and the p38a kinase might
INTRODUCTION play a central role in inflammation [35, 39].
Inhibitors of p38 serve as valuable tools for
Propionibacterium acnes contributes to elucidating the role of that enzyme in
inflammatory acne by stimulating inflammatory responses. SB203580 is an
keratinocytes, sebocytes, and immune cells to inflammatory cytokine inhibitor that blocks
produce multiple proinflammatory cytokines both p38a and p38b kinase activities [37].
such as interleukin (IL)-1a, tumor necrosis SCIO-469 is a relatively selective inhibitor of
factor (TNF)-a, and chemokines (e.g., IL-8), the a isoform of p38 [40]. Topical
which, in turn, attract neutrophils and administration of SCIO-469 reduced acute skin
mononuclear cells to the pilosebaceous unit inflammation in guinea pigs [41].
[1–8]. P. acnes can also directly cause tissue p38 regulates the production of
injury [9–13]. In addition, P. acnes can activate inflammatory mediators that stimulate
inflammasomes and induce IL-1b secretion leukocyte recruitment and activation [42].
from monocytes [14, 15]. Toll-like receptors Many of these mediators (e.g., IL-1, TNF-a, and
(TLRs) and nuclear factor kappa B (NF-jB) are IL-8) are secreted by keratinocytes in response
involved in P. acnes-induced inflammation [4, toP.acnes[5–7].Thus,thepurposeofthisstudy
16–19]. Microbial products and cytokines act was to evaluate whether the P. acnes-induced
through the Toll-like, IL-1, and TNF receptor proinflammatory cytokine release is mediated
families to activate mitogen-activated protein by P. acnes-induced activation of p38 mitogen-
kinases (MAPKs), leading to the activation of activated protein kinase (p38 MAPK or p38) in
NF-jB [20–25]. Very few studies have human keratinocytes.
investigated which MAPK may mediate the
proinflammatory response [26, 27]. METHODS
There are three major groups of MAPKs: the
extracellularsignal-relatedkinases(ERK1/2),the p38 Inhibitors
c-Jun N-terminal kinases (JNK1/2), and the p38
MAPKs (a, b, c, and d) [20, 28–36]. ERKs are SCIO-469 was provided by Scios Inc. (Fremont,
predominantly activated by mitogenic factors, CA, USA) [40]. SB203580 was purchased from
while JNK and p38 are preferentially activated Sigma (St. Louis, MO, USA).
DermatolTher(Heidelb)(2015)5:53–66 55
Cell Culture phospho-p38andthatofhealthyskinwaschosen
withno/weak(nocolor/lightbrowncolor)nuclear
Normal human neonatal epidermal staining of phospho-p38 in follicular and
keratinocytes were obtained from Cascade perifollicularepidermalkeratinocytes.
Biologics (Portland, OR, USA) and maintained
in keratinocyte growth medium (KGM) Human Epidermal Equivalents
(Cascade Biologics, Inc., Portland, OR, USA).
All experiments were performed between Human epidermal equivalents were purchased
passages 2–4 and at 70–80% cell confluence. from MatTek Company (Asland, MA, USA).
KGM was used as antibiotic free in all the Upon receipt, human epidermal equivalents
experiments described in this paper. were incubated in EPI-100-ASY antibiotic-free
MatTek assay medium overnight and then
Propionibacterium acnes changed to KGM for another overnight
incubation before topical treatment.
Propionibacterium acnes strain ATCC 11828 was
obtained from American Type Culture Enzyme-Linked Immunosorbent Assay
Collection (Manassas, VA, USA). Bacteria were (ELISA)
grown for 48–72h to reach stationary phase.
Live bacteria were then harvested and re- The protocol was adapted from Graham et al.
suspended in KGM to achieve 108CFU/mL for [14]withmodifications.Inbrief,humanprimary
in vitro experiments. keratinocytes were pre-incubated for 1h with
either p38 inhibitors or with vehicle, and the
Tissue Specimens treatmentswerestopped.Thekeratinocyteswere
then exposed to stationary-phase P. acnes (at a
Five human acne lesional biopsies (three ratio of P. acnes to keratinocytes of 50:1) and
papules, one pustule, and one cystic acne) and wereco-culturedfor16hat37(cid:3)C.Asforhuman
five healthy scalp/neck skin were kindly epidermal equivalents, they were topically
provided by Drs. Jack L. Arbiser (Department treated with either vehicle or the p38 inhibitors
ofDermatology,EmoryUniversity,Atlanta,GA, for 2h, followed by changing the medium to
USA) and James J. Leyden (Department of KGM in either absence or presence of live
Dermatology, University of Pennsylvania, PA, stationary-phase P. acnes at 108CFU/mL for
Philadelphia, USA) with informed consent. 16h. After incubation, the supernatants were
Five-micrometer-thick sections of biopsies were analyzedusingBeadlyte(cid:4)humanmulti-cytokine
processed for immunostaining with rabbit anti- ELISA beadmasterTM Assays (Upstate Cell
human phospho-p38 MAPK (Thr180/Tyr182) Signaling Solution, Lake Placid, NY, USA).
antibody (Cell Signaling Technology Inc., Results are presented as the mean±SD of eight
Danvers, MA, USA) [43]. replicate wells for keratinocytes or of two
The immunohistochemical assessment was equivalents, in which triplicates were collected
done by visually assessing the intensity of the from each equivalent. Half-maximal inhibitory
signal(browncolor)inacnelesionsvshealthyskin. concentrations (IC ’s) were calculated using
50
Arepresentativeimageofacnelesionswaschosen SigmaPlot Version 9.0 (Csystat Software Inc.,
withstrong(darkbrowncolor)nuclearstainingof San Jose, CA, USA).
56 DermatolTher(Heidelb)(2015)5:53–66
Lactate dehydrogenase (LDH) assays (G- andhighlysignificantifp\0.01(**)orp\0.005
Biosciences, St. Louis, MO, USA) were (***).
performed in all the supernatants to ensure no
cytotoxicity occurred in the cells under the
Compliance with Ethics Guidelines
experimental conditions.
All procedures followed were in accordance
Western Analysis
with the ethical standards of the
responsible committee on human
Activation of p38, ERK1/2, and JNK in human
experimentation (institutional and national)
primary keratinocytes or epidermal
and with the Helsinki Declaration of 1964,
equivalents was evaluated by Western blot
as revised in 2013. Informed consent was
using phospho-specific antibodies, and was
obtained from all patients for being included
normalized to the total level of the relevant
in the study.
proteins using anti-p38, anti-ERK1/2, and
anti-JNK1/2 antibodies. Antibodies for
RESULTS
phospho-p38 (Thr180/Tyr182), p38,
phospho-p44/42 or ERK1/2 (Thr202/Tyr204),
Activation of p38 in Human Acne Lesions
p44/42, phospho-SAPK/JNK (Thr183/Tyr185),
SAPK/JNK, as well as goat anti-rabbit IgG HRP
Phospho-p38 expression was detected in all
(horseradish peroxidase) and horse anti-
five acne tissue samples with a nuclear
mouse IgG HRP secondary antibodies were
localization. A representative image of
obtained from Cell Signaling Technology
healthy skin shows mainly diffuse
(Beverly, MA, USA). All other reagents for
cytoplasmic staining of phospho-p38 in both
Western blot were purchased from Sigma (St.
epidermal and dermal cells (Fig.1a), and weak
Louis, MO, USA), and blotting equipment was
nuclear staining in some hair follicle cells
purchased from Bio-Rad (Hercules, CA, USA).
(Fig.1b). In contrast, markedly increased level
The separated proteins were visualized by an
of phospho-p38 was observed in the
ECL kit according to the manufacturer’s
inflammatory acne lesion (Fig.1c). The
protocol (GE Healthcare Bio-Sciences Co.,
phospho-p38 was mostly confined to the
Piscataway, NJ, USA) and quantitated by
nuclei of follicular and perifollicular
densitometric analysis using a Kodak Gel
epidermal keratinocytes (Fig.1d).
Logic 100 Imaging System and Kodak 1D
Imaging Software (Eastman Kodak Co., New
Haven, CT, USA). Propionibacterium acnes Induces a Time-
Dependent Activation of MAPKs
Statistics in Keratinocytes
Statistical analyses were performed using two- Stationary-phase P. acnes stimulated rapid
tailed Student’s t tests with unequal variances (within 5min) phosphorylation of both p38
(Microsoft Office Excel 2007; Microsoft, (phospho-p38)andERK1/2(phospho-ERK1/2),
Redmond, WA, USA). Differences were with peaks at 15min(Fig.2a).There was up to
considered statistically significant if p\0.05 (*), asevenfoldincreaseinphosphorylationofp38
DermatolTher(Heidelb)(2015)5:53–66 57
Fig.1 p38 is activated in human acne lesions. a Healthy is found in follicular and perifollicular epidermis and
human skin shows diffuse cytoplasmic staining in both dermisofarepresentativehumaninflammatoryacnelesion.
epidermis and dermis. b High magnification insert of d High magnification insert of c demonstrates a solid dot
a illustratesweak nuclearstaining insome hairfolliclecells pattern of nuclear staining phospho-p38. Bar 100lm
ofahealthyskin.cStrongnuclearstainingofphospho-p38
and ERK1/2, compared to untreated (Fig.2b, Propionibacterium acnes-Induced
c). An additional activation peak of phospho- Phospho-p38 and Secretion of Cytokines
ERK1/2 occurred between 2 and 4h are Suppressed by SB203580
(approximately sixfold increase; Fig.2a, c). P.
acnes also induced relatively slow but PretreatmentofthekeratinocyteswithSB203580
sustained phosphorylation of JNK1/2 at 10 and 25lM reduced P. acnes-induced
(phospho-JNK1/2); the maximum increase phospho-p38tobasal levels(Fig.3a).Stationary-
was approximately fourfold between 2 and phaseP.acnesstimulatedtheproductionofTNF-a
8h (Fig.2a, d). These results show that viable (Fig.3b) and IL-8 (Fig.3c). Pretreatment of
P. acnes induced the activation of multiple keratinocytes with SB203580 significantly
MAPK pathways in cultured human suppressed P. acnes-induced secretion of TNF-a
keratinocytes. andIL-8(Fig.3b,c,bothp\0.005).
58 DermatolTher(Heidelb)(2015)5:53–66
a Time (min) 0 1 5 10 15 30 60 120 240 480
b
8
o 7 p-p38
y t 6
nsitol 5
dentr 4
ve co 3
ati 2
Rel 1
0
0 1 5 10 15 30 60 120 240 480
Time (min)
c
10
o p-ERK1/2
y t 8
nsitol 6
dentr
ve co 4
ati 2
el
R
0
0 1 5 10 15 30 60 120 240 480
Time (min)
d 5
y to 4 p-JNK1/2
nsitol 3
ve decontr 2
ati 1
el
R
0
0 1 5 10 15 30 60 120 240 480
Time (min)
Fig.2 Propionibacterium acnes induces activation of mul- phosphorylated MAPKbandwasnormalizedtoitsloading
tiple MAPK pathways in cultured human keratinocytes. control, and then compared to time 0. MAPK mitogen-
a Western blots show time-dependent induction of activated protein kinase, ERK extracellular signal-related
phospho-p38 (p-p38), phospho-ERK1/2 (p-ERK1/2) and kinases, JNK c-Jun N-terminal kinases
phospho-JNK1/2 (p-JNK1/2). b–d The density of each
DermatolTher(Heidelb)(2015)5:53–66 59
Fig.3 Propionibacterium acnes-induced phospho-p38 and b–c SB203580 inhibits the secretion of TNF-a (b) and
cytokine release in keratinocyte cultures was inhibited by a IL-8 (c). Data are expressed as mean±SD. p\0.05 (*),
p38a/b inhibitor, SB203580. a Western blots show that p\0.01 (**), p\0.005 (***). TNF tumor necrosis factor,
SB203580 inhibits P. acnes-induced phospho-p38. PA Propionibacterium acnes, Veh vehicle
Propionibacterium acnes-Induced upregulation of P. acnes induced either
Activation of p38, but not ERK1/2 phospho-ERK1/2 or phospho-JNK1/2 (Fig.4a).
and JNK1/2, and Inflammatory Cytokine/ Stationary-phase P. acnes stimulated the
Chemokine Secretion in Keratinocytes is production of TNF-a (Fig.4b, p\0.005), IL-8
Dose-Dependently Inhibited by SCIO-469 (Fig.4c, p\0.005), and IL-1a (Fig.4d,
p\0.005). Pretreatment of keratinocytes with
Pretreatment of the keratinocytes with SCIO- SCIO-469 dose-dependently inhibited P. acnes-
469 inhibited P. acnes-induced phospho-p38 in induced TNF-a secretion with an IC of 79nM
50
a dose-dependent fashion with a 50% (Fig.4b).SCIO-469alsosignificantlysuppressed
inhibition at 16–80nM, and complete IL-8 and IL-1a secretion (Fig.4c, d), but the
inhibition was observed between 0.4 and 2lM degreeofsuppressionwaslessforIL-8andIL-1a
(Fig.4a). There was no inhibition but than that observed for TNF-a.
60 DermatolTher(Heidelb)(2015)5:53–66
Fig.4 Propionibacteriumacnes-inducedphosphorylationof phospho-JNK1/2 (p-JNK1/2). b–c SCIO-469 inhibits the
p38, but not of ERK1/2 or JNK1/2, and the cytokine secretion of TNF-a (b), IL-8 (c), and IL-1a (d). Data are
release was inhibited by a p38a inhibitor, SCIO-469, in expressed as mean±SD. p\0.05 (*), p\0.01 (**),
humankeratinocytes.aWesternblotsshowthatSCIO-469 p\0.005 (***). ERK extracellular signal-related kinases,
inhibits P. acnes-induced phospho-p38 (p-p38) dose-de- JNKc-JunN-terminalkinases,TNFtumornecrosisfactor,
pendently, but not of phospho-ERK1/2 (p-ERK1/2) or IL interleukin, PA Propionibacterium acnes
DermatolTher(Heidelb)(2015)5:53–66 61
Topical SCIO-469 Inhibits P. acnes- p38 dose-dependently (Fig.5a) as compared to
Induced Phospho-p38 and Secretion P. acnes-induced plus vehicle-treated control
of Cytokines in Human Epidermal (Veh?PA). In addition, topical SCIO-469
Equivalents suppressed P. acnes-induced secretion of both
TNF-a and IL-8 significantly (p\0.005) at 20
Viable P. acnes induced both phospho-p38, and and 100lM (Fig.5b, c). As expected, viable P.
secretion of TNF-a and IL-8 from the acnes also induced secretion ofIL-1ainthe skin
equivalents, compared to the control without equivalents.InhibitionofIL-1abytopicalSCIO-
P.acnes(Veh-PA,Fig.5a,b).TopicalSCIO-469at 469 was also observed, but with no obvious
4–100lM inhibited P. acnes-induced phospho- dose-response curve (data not shown).
DISCUSSION
Inthisstudy,p38MAPKwashighlyactivatedin
the inflammatory acne lesions compared to
normal skin. Using small molecule p38
inhibitors, it was further demonstrated that
these compounds not only suppressed P. acnes-
induced phosphorylation of p38 in vitro, but
also decreased P. acnes-induced
proinflammatory cytokine and chemokine
production in keratinocytes in vitro. These
data suggest that activation of p38 by P. acnes
may be one of the contributors to the clinical
development of inflammatory acne.
The current study was a pilot investigation
on the histological and histochemical skin
changes showing there was activation of p38
signaling in inflammatory acne lesions. A
limitation of the current study was the small
numberofacnebiopsiesthatcould beobtained
and evaluated. Obtaining biopsies of acne
lesions was challenging which limits the
extrapolation of the results. In addition, this
Fig.5 Propionibacterium acnes-induced phospho-p38 and
study evaluated p38 only in inflammatory acne
cytokine release was inhibited by a p38a inhibitor,SCIO-
lesions; it could be interesting to examine p38
469, in human epidermal equivalents. a Western blot
shows that SCIO-469 suppresses P. acnes-induced phos- activation in early lesional samples such as
pho-p38 (p-p38) dose-dependently. b–c SCIO-469 in- comedones.
hibits the secretion of TNF-a (b) and IL-8 (c). Data are
ExposureofkeratinocytestoP.acnesinduced
expressed as mean±SD. p\0.05 (*), p\0.01 (**),
the activation of multiple MAPK pathways.
p\0.005 (***). PA Propionibacterium acnes, TNF tumor
necrosis factor, IL interleukin SB203580, a p38a/b inhibitor, inhibited
62 DermatolTher(Heidelb)(2015)5:53–66
P. acnes-induced phospho-p38. A second p38 staining in follicular and perifollicular
pharmacological inhibitor, SCIO-469, epidermal keratinocytes, suggesting
specifically inhibited P. acnes-induced translocation of activated p38 to the nuclei.
phospho-p38 in a dose-dependent fashion, The finding is similar to the localization of
suggesting the major isoform activated by P. activated NK-jB and activator protein-1 (AP-1)
acnes in keratinocytes is p38a. However, when in inflammatory acne lesions [17]. The
phospho-p38 was blocked by SCIO-469, a proliferative effect could be due to the ability
concomitant upregulation of phospho-ERK1/2 of P. acnes to activate p38 by stabilizing IL-8
and phospho-JNK1/2 was observed. This mRNA, and subsequently to upregulate IL-8
observation was consistent with previous secretion resulting in keratinocyte proliferation
findings [40, 44]. Similar data were also [52–55]. Both live and heat-killed P. acnes can
reported in a wound-healing model and a induce phosphorylation of heat shock protein
conditional p38a knock-out mouse model 27,adownstreamcellularstressmediatorofp38
[45, 46]. [26]. Although our study only focused on acne
Propionibacterium acnes stimulates the lesional skin and non-lesional healthy skin,
secretion of proinflammatory cytokines and previous studies have reported that non-
chemokines such as IL-1, TNF-a, and IL-8 in lesional acne skin adjacent to acne lesions
cultured keratinocytes. IL-1a induces (perilesional) showed overexpression of
hypercornification of the infundibulum proinflammatory cytokines (IL-1a) and
ex vivo, and was detected in the majority of increased immune cells, but did not exhibit
open comedonesof acne patients [47–49]. TNF- hyperproliferation or abnormal differentiation
a may drive the early stage of inflammatory ofthefollicularepithelium[56].Itisinteresting
acne lesions [5]. IL-8 is a potent chemotactic to speculate that the presence of inflammation
agent for neutrophils, and both IL-8 and alone is not sufficient to produce acne but
neutrophils are commonly found in high perhaps, P. acnes-induced inflammatory
levels in inflammatory acne lesions [17, 50]. signaling is an initiating event in comedone
IL-8-induced chemotaxis of neutrophils may be formation; however, to produce clinical acne
activated by endogenous or exogenous (e.g., lesions may require a number of P. acnes-
bacterial) factors to release lysosomal enzymes induced signaling pathways in keratinocytes
that can degrade the follicular epithelium, that affect inflammatory responses, cell
leading to further inflammation [51]. In proliferation, and hyperkeratinization.
lipopolysaccharide (LPS)-stimulated human
monocytes, p38a MAPK was the molecular
target for the biosynthesis of TNF-a and IL-1 CONCLUSION
[20, 21]. Data presented here not only confirm
theincreasedproductionofTNF-a,IL-8,andIL- This study shows an increase in phospho-p38 in
1 by keratinocytes upon stimulation with P. biopsies of acne lesions predominantly in
acnes, but also demonstrated the involvement follicularandperifollicularkeratinocytes.P.acnes
of p38a MAPK in this process. induces inflammatory responses in cultured
Moreover, the biopsies from clinically keratinocytes and skin epidermal equivalents.
diagnosed inflammatory acne lesions showed a p38 MAP kinase inhibitors reduce P. acnes-
marked increase of phospho-p38 nuclear induced inflammation in vitro. These findings,