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ORIGINAL ARTICLE

Histone H4 Is a Major Component of the


Antimicrobial Action of Human Sebocytes
Dong-Youn Lee1,4, Chun-Ming Huang1, Teruaki Nakatsuji1, Diane Thiboutot2,
Sun-Ah Kang3, Marc Monestier3 and Richard L. Gallo1

Antimicrobial peptides, such as cathelicidin and b defensins, directly kill microbes and have been detected in
human sebaceous glands and cell lines. Despite the presence of several such peptides, the apparent abundance
of these is insufficient for direct killing of most skin pathogens. In this study, we sought to determine which
molecules provide the majority of antimicrobial peptide activity in human sebocytes. Acid-soluble protein
extracts of SEB-1 sebocytes were separated by reverse-phase high-performance liquid chromatography and
were assayed for their capacity to inhibit the growth of Staphylococcus aureus. Antimicrobial activity was
isolated in a single major fraction and identified to be histone H4 by mass spectrometry and western blot
analysis. The importance of histone H4 in the antimicrobial activity of sebocytes was confirmed by a specific
neutralizing antibody and by direct demonstration that recombinant histone H4 had antimicrobial activity
against S. aureus and Propionibacterium acnes. In addition, histone H4 enhanced the antimicrobial action of
free fatty acids in human sebum. Taken together, these results indicate that the release of histone H4 by
holocrine secretion from the sebaceous gland may play an important role in innate immunity.
Journal of Investigative Dermatology (2009) 129, 24892496; doi:10.1038/jid.2009.106; published online 18 June 2009

INTRODUCTION and proteins in the skin also show antimicrobial activity,


Antimicrobial peptides are crucial components of the innate including proteinase inhibitors, chemokines, and neuropep-
immune system that protects against microbial infection tides (Braff et al., 2005).
(Zasloff, 2002). In part, acting as natural antibiotics, The sebaceous gland is a major appendage and exocrine
antimicrobial peptides have a distinct and overlapping gland in the skin consisting of specialized epithelial cells
antimicrobial activity against a variety of microbial pathogens named sebocytes (Thody and Shuster, 1989; Thiboutot,
(Bals, 2000; Nagaoka et al., 2000). They are produced 2004). Human sebaceous glands are distributed in all areas
constitutively or can be induced in a variety of types, of the skin except in that of the palms and soles. Although the
including phagocytes and epithelial cells (Gallo et al., 1994; sebaceous gland produces and secretes lipid-rich sebum onto
Ganz, 2002), and they can directly kill a broad spectrum of the external skin surface by the holocrine rupture of mature
bacteria, fungi, and viruses. Their role is particularly sebocytes, the fundamental functions of this gland have been
important in the skin where they play an important role in only partly understood (Zouboulis, 2003). These functions
first-line defense against infection (Izadpanah and Gallo, include the hypothesis that the sebocyte provides an innate
2005). In the keratinocyte, cathelicidins and b defensins are immune defensive function. This has been supported by
major families of antimicrobial peptides (Frohm et al., 1997; observations that antimicrobial peptides are produced by
Ali et al., 2001). In addition, however, many other peptides these cells. For example, human b defensins (HBD)-1 and -2
were found in human pilosebaceous units by in situ
1
hybridization and immunohistochemistry (Chronnell et al.,
Division of Dermatology, University of California San Diego and VA San
Diego Healthcare Center, San Diego, California, USA; 2Department of 2001). An HBD-2 mRNA expression was induced in SZ95
Dermatology, Milton S. Hershey Medical Center, Pennsylvania State sebocytes by Propionibacterium acnes (P. acnes), a predo-
University College of Medicine, Hershey, Pennsylvania, USA; 3Department of minant organism in pilosebaceous units (Nagy et al., 2006).
Microbiology & Immunology, Temple University School of Medicine,
Psoriasin (S100A7) has been detected in sebaceous glands by
Philadelphia, Pennsylvania, USA and 4Department of Dermatology, Samsung
Medical Center, Sungkyunkwan University School of Medicine, Seoul, South immunohistochemistry (Glaser et al., 2005). Recently, we
Korea reported that human sebocytes express cathelicidin antimi-
Correspondence: Dr Richard L. Gallo, Division of Dermatology, University of crobial peptides and can act to kill P. acnes (Lee et al., 2008).
California San Diego, VA San Diego Healthcare Center, 3350 La Jolla Village Moreover, sebaceous lipids exhibit antibacterial activity
Drive, Mail Code 151, San Diego, California, 92161, USA.
E-mail: rgallo@ucsd.edu
(Wille and Kydonieus, 2003; Georgel et al., 2005). These
findings suggested that the human sebaceous gland may play
Abbreviations: HPLC, high-performance liquid chromatography; MS, mass
spectrometry an important role in the innate immunity of the skin through
Received 4 November 2008; revised 8 February 2009; accepted 24 February the expression of antimicrobial peptides and specific lipids.
2009; published online 18 June 2009 However, the measured concentration of the previously

& 2009 The Society for Investigative Dermatology www.jidonline.org 2489


D-Y Lee et al.
Histone H4 is an Antimicrobial of Sebocytes

defined antimicrobial peptides in the extracts of sebocytes at approximately 11 kDa in the active fraction was found to
was lower than the concentration necessary for antimicrobial be interesting. To identify this molecule, tryptic peptide
activity. Thus, although multiple antimicrobial peptides have digests were analyzed by Nano liquid chromatography-mass
been detected in sebocytes, it remains unclear which are spectrometry (MS). Histones, H2A, H2B, and H4, were
functionally important to immune defense. Here, we carried identified with H4 being most abundant (Table 1).
out an unbiased functional screening of proteins derived from To confirm these results, each of the HPLC fractions was
sebocytes for antimicrobial activity. On the basis of this evaluated by western blot with an anti-histone antibody
screening, we identified histone H4 as the predominant (BWA-3). BWA-3 is a monoclonal antibody that reacts with a
peptide active against Staphylococcus aureus (S. aureus) and common sequence in the amino-terminus of histones H2A
P. acnes. and H4 (Monestier et al., 1993). In fraction 13, one strong
band was seen at 11 kDa, which corresponded to the
RESULTS expected size of histone H4 (Figure 2a) and to the unique
Purification of antimicrobial activity from cultured human protein predicted to have antimicrobial activity in Figure 1c.
sebocytes In fraction 12, one strong band was observed at 13 kDa,
To identify antimicrobial molecules produced by the sebac- which corresponded to the expected size of histone H2A
eous gland, SEB-1 sebocytes were subjected to a variety of (Figure 2a). An additional western blot analysis with a
initial extraction methods including chloroform, detergent, different antibody that specifically reacts with acetyl histone
and aqueous extraction techniques. Only acid extracts, H4 confirmed that the band in fraction 13 is H4 (Figure 2b).
predominantly representing the acid-soluble protein fraction Evaluation of the total acid-soluble extracts of SEB-1 by
of the cell, were antimicrobial in the solution-killing assay western blot with the anti-histone antibody (BWA-3) further
against S. aureus DmprF, a well-defined S. aureus strain confirmed the presence of the two histone bands at 11 and
selected for its sensitivity to cationic antimicrobial peptides. 13 kDa, corresponding to histones H4 and H2A (Figure 2c).
These extracts were subsequently separated by reverse-phase Histone H4 was much more abundant than histone H2A
high-performance liquid chromatography (RP-HPLC) in the total extract. A similar result was obtained in the
(Figure 1a) and by the bactericidal activity of the HPLC acid-soluble fraction of the SEB-1 debris released into the
fractions examined against S. aureus by limiting dilution. In culture medium, also showing two bands, with the majority
this screening, only a single fraction (fraction 13) was being histone H4 (Figure 2c). To further confirm histone H4
confirmed to have an antibacterial activity (Figure 1b). expression in human sebaceous glands, histological sections
of normal human scalp tissue were examined for histone H4
Identification of Histone H4 as an antimicrobial peptide expression by immunohistochemistry with BWA-3. As
As predicted by the initial HPLC tracing, each of the fractions expected for this essential component of the chromosome,
separated from sebocyte extracts contained multiple protein histone H4 was detected in the nuclei of the sebocytes and in
bands (Figure 1c). On the basis of the findings of the those of all other cells within the biopsy (data not shown).
antimicrobial activity and the SDS-PAGE, one band migrating Thus, on the basis of the results of functional HPLC analysis,

HPLC fractions
mAU %B 9 10 11 12 13 14 15 16 17 Control
Ten fold dilutions

80
3,000
Acetonitrile concentration
OD 214 nm

60
2,000

40 HPLC fractions
(kDa) Marker 9 10 11 12 13 14 15
1,000 36

20 22
16

0 0 6
20.0 30.0 4

Figure 1. Purification of the antimicrobial activity from extracts of cultured sebocytes. (a) RP-HPLC was carried out to separate antimicrobial peptides from
acid-soluble extracts of SEB-1 sebocytes. A plot of absorbance at 214 nm is indicated on the left y axis and that of the concentration of acetonitrile used for
elution on the right y axis. (b) The antimicrobial activity of the HPLC fractions was evaluated against S. aureus DmprF by solution-killing assay. Growth of
bacterial colonies on agar after incubation with fractions obtained by RP-HPLC separation is shown with limiting dilutions from top to bottom. A single fraction
(fraction 13) showed maximal antimicrobial activity. (c) HPLC fractions were subjected to SDS-PAGE and stained with Coomassies blue. Arrow in the active
fraction indicates a unique protein band that was further analyzed by mass spectrometry.

2490 Journal of Investigative Dermatology (2009), Volume 129


D-Y Lee et al.
Histone H4 is an Antimicrobial of Sebocytes

Table 1. Proteins identified by mass spectrometry HPLC fractions


from sebocyte antimicrobial fraction (kDa) H4 10 11 12 13 14 15
Identified Accession Sequence
protein no./database Peptide sequence coverage (%) 16 H2A
Histone H4 NP_778224.1/ ISGLIYEETR 63.1 H4
6
NCBI TLYGFGG
DAVTYTEHAK
KTVTAMDVVYALK HPLC fractions

TVTAMDVVYALK (kDa) 10 11 12 13 14 15
VFLENVIR
16
RISGLIYEETR
Acetyl H4
DNIQGITKPAIR 6
DNIQGITK
DNIQGITKPAIRR
SEB-1
NP_003538.1/ ILGLIYEETRR 19.4
(kDa) H4 Cells Debris
NCBI ILGLIYEETR
DNIQGITK
16 H2A
Histone H2B NP_778225.1/ AMGIMNSFVNDIFER 27
H4
NCBI KESYSIYVYK
LLLPGELAK 6
NP_066407.1/ KESYSVYVYK 27
NCBI LLLPGELAK Figure 2. Identification of histone H4 in antimicrobial fractions purified
AMGIMNSFVNDIFER from cultured sebocytes. (a) Western blot analysis with anti-histone antibody
(BWA3, specific for histones H2A and H4) was carried out on HPLC fractions
Histone H2A NP_002097.1/ ATIAGGGVIPHIHK 23.4 shown in Figure 1. In fraction 13, a single strong band was observed at
NCBI AGLQFPVGR 11 kDa, which corresponded to the expected mass of histone H4. In fraction
12, a single strong band was observed at 13 kDa, which corresponded to the
HLQLAIR
expected mass of histone H2A. The lane marked as H4 was loaded with
recombinant histone H4 used as a positive control. (b) Western blot analysis
with the specific anti-acetyl histone H4 antibody showed a single band at
11 kDa only in fraction 13. (c) Acid-soluble extracts of SEB-1 sebocytes and
the culture medium containing SEB-1 sebocyte debris was evaluated by
Coomassie staining, mass spectrometry, and western blot western blot with an anti-histone H2A and H4 antibody (BWA-3). Two bands
analysis, histone H4 was identified as a top candidate were observed at 11 and 13 kDa, which corresponded to histones H4 and
responsible for the antimicrobial activity in fraction 13. H2A. H4, recombinant histone H4, was used as a positive control.

Histone H4 has direct antimicrobial activity against S. aureus


and P. acnes histone H4, but oleic acid at this concentration killed all of
To confirm whether histone H4 can kill bacteria found on the the bacteria. Thus, as expected, free fatty acids present on the
skin, bactericidal assays were carried out using a purified skin are inherently antimicrobial, but will also enhance the
recombinant histone H4 protein against relevant skin patho- antimicrobial potency of peptides such as histone H4
gens such as S. aureus or P. acnes. Bacteria were incubated released by the sebocyte.
for 3 hours at 371C in an assay buffer consisting of a 10 mM
sodium phosphate buffer (pH 6.5) and 100 mM NaCl. Histone Histone H4 is a major component of the antimicrobial activity
H4 showed bactericidal activity against both S. aureus DmprF of sebocyte extracts
(25 mg ml1) and P. acnes (12.5 mg ml1) in a dose-dependent Having found that histone H4 is a possible antimicrobial
manner compared with the control (Figure 3). Furthermore, protein, we next sought to determine the relative contribution
as histone H4 is released from sebocytes along with lipids, of histone H4 to the functional antimicrobial activity
which are metabolized into free fatty acids with antimicrobial observed from sebocytes. The specific histone antibody
activity, the combined antimicrobial activity of histone H4 in (BWA-3) described earlier (Kim et al., 2002) was used in a
the presence of free fatty acids was examined. Lauric acid functional assay in an attempt to block the activity of histones
and oleic acid at low concentrations (1.5 mg ml1) enhanced H4 and H2A in the entire SEB-1 extract. The addition of
the antimicrobial action of histone H4 (Figure 4). At higher BWA-3 to the extracts of SEB-1 sebocytes significantly
concentrations of fatty acids (60 mg ml1), lauric acid inhibited antimicrobial activity against S. aureus DmprF as
completely inhibited S. aureus survival at 12.5 mg ml1 seen by an approximately two logs better growth of bacteria

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D-Y Lee et al.
Histone H4 is an Antimicrobial of Sebocytes

S. aureus mprF P. acnes


(CFU ml1) (CFU ml1)
1.0E+07 1.0E+08
** **
** **
1.0E+06 1.0E+07 **
** **
1.0E+06
1.0E+05
1.0E+05
1.0E+04
1.0E+04

1.0E+03
1.0E+03

1.0E+02 1.0E+02
100 50 25 12.5 Control 100 50 25 12.5 Control

Histone H4 (g ml1) Histone H4 (g ml1)

Figure 3. Recombinant histone H4 kills S. aureus and P. acnes. The antimicrobial activity of recombinant histone H4 was evaluated against S. aureus DmprF
and P. acnes by the solution-killing assay. Bacteria were incubated for 3 hours at 371C in a 10 mM sodium phosphate buffer (pH 6.5) containing 100 mM NaCl.
Recombinant histone H4 showed significant bactericidal activity against S. aureus DmprF at 25 mg ml1 and above and against P. acnes at 12.5 mg ml1 and
above. Data shown are from triplicate determinations (**Po0.01; Students t-test).

S. aureus mprF S. aureus mprF


(CFU ml 1) (CFU ml 1)
1.0E+06 1.0E+06

DMSO DMSO
1.0E+05 LA (1.5) 1.0E+05 LA (60)
OA (1.5) OA (60)
1.0E+04 1.0E+04

1.0E+03 1.0E+03

1.0E+02 1.0E+02

1.0E+01 1.0E+01

1.0E+00 1.0E+00
Control 12.5 25 50 Control 12.5 25 50
Histone H4 (g ml 1) Histone H4 (g ml 1)

Figure 4. Recombinant histone H4 enhances the antimicrobial action of free fatty acids. The antimicrobial activity of histone H4 was evaluated in the presence
of free fatty acids. Lauric and oleic acids (1.5 mg ml1) synergistically exerted antimicrobial effects in combination with histone H4. Lauric acid (60 mg ml1)
exerted a stronger synergy with histone H4, but oleic acid at this concentration killed all bacteria. Data shown are from triplicate determinations (*Po0.05;
Students t-test). DMSO, dimethyl sulfoxide: LA, lauric acid: OA, oleic acid.

in the presence of a sebocyte extract plus BWA-3 compared major antimicrobial protein in the extracts of SEB-1 sebo-
with that with a sebocyte extract plus IgG at a similar cytes. Recombinant histone H4 showed antimicrobial activity
concentration, or with PBS as controls (Figure 5). Thus, the against S. aureus and P. acnes, both potential pathogens of
ability to neutralize the majority of the antimicrobial action of the human pilosebaceous unit. Finally, by selective neutra-
sebocyte extracts with a specific antibody to histone H4 lization of histone, this protein was found to have a major
suggests that histone H4 is the major acid-soluble antimicro- role in the antimicrobial activity of sebocytes. These
bial protein released by the sebocyte. observations suggest that histone H4 in the human sebaceous
gland can contribute to the innate immune defense of the
DISCUSSION skin, providing an additional source of antimicrobial activity
Many studies have shown that antimicrobial peptides are that acts in the defense against infection.
essential elements of epithelial defense, providing a barrier To our knowledge, this is the first report showing that
against infection of the skin and other epithelial organs histones can act as antimicrobial peptides on human skin.
(Zasloff, 2002; Braff et al., 2005). Human sebaceous glands Although histone proteins exist primarily in the nucleus and
may contribute to the defense of the skin through the release are known for their involvement in chromatin formation and
of antimicrobial peptides (Chronnell et al., 2001; Glaser in the regulation of transcription, there is precedence for the
et al., 2005, Lee et al., 2008). In this study, we examined conclusion that histones or histone-derived peptides can have
human sebocytes to identify functionally important antimi- antimicrobial properties (Parseghian and Luhrs, 2006). Other
crobials in human sebaceous glands. In a non-biased activities such as hormone-like activity and endotoxin-
biochemical screening, histone H4 was found to be the neutralizing activity have also been reported for histones

2492 Journal of Investigative Dermatology (2009), Volume 129


D-Y Lee et al.
Histone H4 is an Antimicrobial of Sebocytes

S. aureus mprF et al., 2007). Recently, the post-translationally modified


(CFU ml 1)
1.0E+06 histone H4 was purified from the skin extract of a Japanese
* tree frog, but the protein did not show any antimicrobial
* activity (Kawasaki et al., 2008). Histone H4, as well as H2A,
1.0E+05
H2B, and H3, have shown bacterial LPS-binding abilities
1.0E+04
(Augusto et al., 2003). In addition, as mentioned earlier, the
neutrophil extracellular traps have been observed to react
with antibodies against the histones, H1, H2A, H2B, H3, and
1.0E+03
H4, and may be a part of their antimicrobial function
(Brinkmann et al., 2004). In this study, we showed by
1.0E+02
PBS SEB-1 SEB-1 SEB-1 solution-killing assay that histone H4 contributes to the
+
BWA3
+
IgG
+
PBS
antimicrobial activity of the extracts of SEB-1 sebocytes, and
that this occurs in physiologically relevant ionic conditions
Figure 5. Neutralization of histone H4 in sebocyte extracts removes and against relevant microbes such as S. aureus and P. acnes.
antimicrobial activity. BWA-3 neutralizing antibody was used to treat extracts
To detect histones in HPLC fractions and in the extracts of
of SEB-1 sebocytes. Addition of BWA-3 (400 mg ml1) to the extracts of SEB-1
sebocytes significantly decreased antimicrobial activity against S. aureus
sebocytes, we used a monoclonal anti-histone antibody
DmprF compared with an equal concentration of IgG added to separate (BWA-3). BWA-3 reacts with peptides l20 of histone H2A
aliquots, or aliquots to which an equal volume of PBS was added as controls. and l29 of histone H4 (Monestier et al., 1993). This antibody
Data shown are from triplicate determinations (*Po0.05; Students t-test). was previously also used as a neutralizing antibody (Kim
et al., 2002). As a result, it was relevant to test the addition of
this antibody as a neutralizing antibody to the extracts of SEB-
(Reichhart et al., 1985; Kim et al., 2002). In humans, histone 1 sebocytes, and results show that this significantly decreased
H1, derived from epithelial cells in the gastrointestinal tract, the antimicrobial activity. Although the neutralizing antibody
is active against Salmonella typhimurium (Rose et al., 1998). blocked both histones H2A and H4, the extracts of the
The histones, H2A and H2B, are secreted into the amniotic sebocytes had a higher histone H4 content than a histone
fluid from epithelial cells on the surface of the human H2A content, and antimicrobial activity was not detected in
placenta and they participate in host defenses of the fetus HPLC fractions that contained abundant histone H2A but not
(Kim et al., 2002). Recently, neutrophil extracellular traps histone H4. These results suggested that the antimicrobial
containing histones in human neutrophils were identified as a activity in the extracts of sebocytes was likely because of
novel antimicrobial mechanism (Brinkmann et al., 2004). In histone H4 and not because of histone H2A.
this study, the antimicrobial action of histone is particularly Several antimicrobial peptides are found in sebocytes and
relevant in the setting of the sebaceous gland function, as may work with histone H4 to increase antimicrobial activity.
these cells secrete their contents into the sebum by Earlier, human b defensins-1 and -2 were described in human
disintegration of the entire cell, a process known as holocrine pilosebaceous units, including the sebaceous glands (Chron-
secretion. Thus, it is particularly relevant for sebocytes to use nell et al., 2001). Psoriasin (S100A7) was found in human
histones as a part of their antimicrobial system. The current sebaceous glands (Glaser et al., 2005) and our lab has
data detecting antimicrobial activity for histone H4 in the recently shown that cathelicidin antimicrobial peptides are
extracts of human sebocyte cultures, and in the culture present in cultured human sebocytes and glands (Lee et al.,
medium from these cells, support the conclusion that histone 2008). On the basis of the results of the neutralization of
H4 from human sebaceous glands in vivo is released with antimicrobial activity with an anti-histone H4 antibody, our
sebum onto the surface of the skin, and once released can act data suggest that histone H4 probably plays a larger role as a
as an antimicrobial. direct antimicrobial substance than do the other antimicro-
As major components of the nucleosome structure in bial peptides previously identified in the sebocyte. This
eukaryotic cells, the histones are functionally classified into conclusion is consistent with the approximate concentration
two groups: linker histones (H1), which seal loops of DNA of these antimicrobial peptides compared with their potency
and keep the nucleosome structures condensed in compact to kill bacteria. However, the actual concentration of histone
conformations, and core histones (H2A, H2B, H3, and H4), H4 and other antimicrobial peptides released by human
which form an octameric complex to produce the nucleo- sebaceous glands remains unknown. Nevertheless, on the
some. Most earlier studies have shown that the lysine-rich basis of the known additive or synergistic activity of
histones (H1, H2A, and H2B) and their fragments have antimicrobial peptides (Nagaoka et al., 2000), it is likely that
antimicrobial activity (Hiemstra et al., 1993; Rose et al., these factors work together to provide an immune defense.
1998; Kim et al., 2002; Howell et al., 2003). There have been In addition to antimicrobial peptides in the human
only a few reports on the antimicrobial activities of histone sebaceous gland, sebaceous lipids have been shown to
H4. A mixture of histones H2B and H4 obtained from exhibit an innate antimicrobial activity (Georgel et al., 2005;
hemocytes of Pacific shrimp showed an activity against Clarke et al., 2007). In the sebum, oleic acid predominates,
Gram-positive bacteria (Patat et al., 2004). In human and lauric acid is one of the strongest antimicrobial lipids
meconium, the histones H2A and H4 have been isolated against Gram-positive bacteria, although it is a minor free
and identified by their antimicrobial activity (Kai-Larsen fatty acid (Wille and Kydonieus, 2003; Bodoprost and

www.jidonline.org 2493
D-Y Lee et al.
Histone H4 is an Antimicrobial of Sebocytes

Rosemeyer, 2007). Thus, we chose these free fatty acids for All collected fractions (1.5 ml) were frozen and lyophilized, and
combined effects with histone H4. Histone H4 showed then resuspended in distilled water for antimicrobial assay and
synergistic antimicrobial effects with free fatty acids against immunoassay. The protein concentration was determined using a
S. aureus. These findings suggest that the production of lipids bicinchoninic acid protein assay (Pierce, Rockford, IL), with BSA as a
by the sebaceous gland, and the metabolism to antimicrobial standard.
free fatty acids by resident microbes, may further contribute
to epithelial defense by combining activity with histone H4 Antimicrobial activity assay of HPLC fractions
and the antimicrobial peptides. However, an alternative For screening the antimicrobial activity of the HPLC fractions, a
explanation for the role of histone H4 in the sebaceous gland solution-killing assay was carried out as described earlier (Lee et al.,
is that it may serve a more specialized and focused role in the 2008). Lyophilized HPLC fractions were resuspended in 30 ml of
protection of the gland itself against infection. In this study, distilled water and were then tested against S. aureus DmprF (a gift
histone H4 was shown to kill P. acnes, a normal microflora in from A. Peschel, Microbial Genetics, University of Tubingen,
the pilosebaceous unit. Therefore, sebaceous antimicrobial Tubingen, Germany). This strain of S. aureus was selected for
peptides and lipids may act only under special circum- screening because of its increased sensitivity to cationic peptides.
stances, such as microbial proliferation in the follicle. S. aureus DmprF was grown in a tryptic soy broth and collected in
Taken together, our data suggest that histone H4 is a the log phase. The number of S. aureus DmprF was enumerated by
functionally important antimicrobial peptide released by the applying a conversion factor (2.0  108 bacteria per ml 0.6 OD
holocrine secretion mechanism of human sebocytes. Further, unit at 600 nm). The bacteria (CFU) were suspended in a 10 mM
in vivo studies of the surface expression of histones on human sodium phosphate buffer (pH 6.5) supplemented with 100 mM NaCl,
skin, and mechanistic studies to determine how histone H4 and 5 ml of HPLC fractions were added and incubated at 371C for 3 h.
acts as an antimicrobial peptide, are required to fully After incubation, 10-fold dilutions were prepared and plated
elucidate their role in skin defense. on tryptic soy broth agar and the plates were incubated for 1 day
at 371C.
MATERIALS AND METHODS
Culture of SEB-1 human sebocytes SDS-PAGE
The immortalized human sebaceous gland cell (sebocyte) line, SEB- The HPLC fractions were analyzed for the content of proteins/
1, was grown in a culture medium consisting of DMEM with 5.5 mM peptides by SDS-PAGE. An equal volume of HPLC fractions was
glucose/Hams F-12 3:1, fetal bovine serum 2.5%, adenine prepared by mixing with a sample buffer, and was then denatured
1.8  104 M, hydrocortisone 0.4 mg ml1, insulin 10 ng ml1, epi- at 951C for 5 minutes. Electrophoresis was carried out on 16%
dermal growth factor 3 ng ml1, and cholera toxin 1.2  1010 M at Tris-tricine gels (NuSep, Austell, GA) with running buffer for 2 hours
371C in a humidified atmosphere containing 5% CO2 in air at 100 V. To visualize the protein/peptide bands, the gel was
(Thiboutot et al., 2003). stained with Coomassie solution according to the manufacturers
instructions.
Extraction of acid-soluble proteins from SEB-1 sebocytes
SEB-1 sebocytes were cultured to confluence in 75 cm2 culture Mass spectrometry analysis
flasks. After washing with PBS, SEB-1 sebocytes were extracted in For protein identification, one band of interest in the active fraction
1 M HCl-1% trifluoroacetic acid. The extracts of the SEB-1 sebocytes was cut out of the Coomassie stained gel and enzymatically digested
in the acid were centrifuged at 12,000 r.p.m., and the supernatant with trypsin (0.2 mg, 25 mM Tris, 4 M urea, pH 7.8) at 371C overnight.
was collected. The supernatant was then frozen and lyophilized. In Tryptic peptide digests were analyzed by Nano liquid chromato-
addition, the debris of the SEB-1 sebocytes in the culture medium graphy-mass spectrometry as described earlier (Nakatsuji et al.,
was centrifuged and extracted in the same way as the extraction of 2008). The automated Nano liquid chromatography-mass spectro-
the SEB-1 sebocytes. metry setup consisted of an Agilent 1200 Nano 2D LC system, a
switch valve, a C18 trap column (Agilent, Santa Clara, CA), and a
Separation of antimicrobial peptides capillary reverse-phased column (10 cm in length, 75 mm i.d.)
Antimicrobial peptides were separated from the acid extracts of the packed with 5 mm C18 AQUASIL resin with an integral spray tip
SEB-1 sebocytes by a previously described method with slight (Picofrit, 15 mm tip, New Objective, Woburn, MA). A reverse-phase
modifications (Murakami et al., 2004). The lyophilized material of LC, directly linked to a Bruker Daltonics HCTultra PTM system mass
the acid-soluble protein extracts of the SEB-1 sebocytes was spectrometer (Bruker Daltoniks, Bremen, Germany), was carried out
dissolved in 10% acetonitrile-0.1% trifluoroacetic acid. Peptide using a linear gradient elution from buffer A (H2O plus 0.1% formic
separation was carried out using an AKTA purification system acid) to 50% buffer A plus 50% buffer B (acetonitrile plus 0.1%
(Amersham Pharmacia Biotech, Piscataway, NJ) on a Sephasil formic acid) in 100 minutes. The setup was operated in the data-
peptide C18 column (12 mm, ST 4.6/250; Amersham Pharmacia dependent mode. Data on the four strongest ions above an intensity
Biotech). The acid-soluble protein extracts of the SEB-1 sebocytes of 2  105 were collected with dynamic exclusion enabled, and the
were separated by reverse-phase high-performance liquid chroma- collision energy was set at 35%. The MS/MS spectra were extracted
tography (RP-HPLC) after column equilibration in 0.1% trifluoroa- using a default value by BioTools 3.1 (Bruker Daltoniks). All MS/MS
cetic acid at a flow rate of 2 ml min1. Elution was carried out using spectra were analyzed using in-house Mascot 2.1 server (Matrix
gradients of 025% and 2560% acetonitrile for 5 or 25 minutes. The Science, London, UK) for protein identification. Mascot was
column effluent was monitored at 214, 230, and 280 nm. established to search the human database (downloaded from the

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D-Y Lee et al.
Histone H4 is an Antimicrobial of Sebocytes

NCBI website http://www.ncbi.nlm.nih.gov/) containing protein (400 mg ml1) or with the same concentration of control IgG or
sequences in both forward and reverse orientations using trypsin as PBS for 2 hours at room temperature and analyzed for antimicrobial
a digestion enzyme. activity against S. aureus DmprF using the solution-killing assay.
To evaluate the combined antimicrobial activity of histone H4
Western blot analysis with free fatty acids, a solution-killing assay was carried out. Two
The fractions separated by HPLC were evaluated by western blot free fatty acids (Sigma, St. Louis, MO), which were dissolved in
analysis. First, SDS-PAGE was carried out as described above and then dimethyl sulfoxide (DMSO), were tested against S. aureus DmprF.
transferred onto a polyvinylidene difluoride membrane (Immobilon-P; The bacteria were exposed to a range of recombinant histone H4
Millipore, Danvers, MA). For a positive control, the recombinant peptide concentrations (50 mg ml1, 25 mg ml1, and 12.5 mg ml1) in
histone H4 peptide (Biolabs, New England) was used. The membrane the PBS buffer in the presence of lauric or oleic acid (1.5 mg ml1 and
was treated with a blocking solution for 30 minutes at room 60 mg ml1) at 371C for 3 hours. DMSO [5% (v v1)] was used as a
temperature, and then a mouse monoclonal anti-histone antibody negative control. After incubation, 10-fold dilutions were prepared
(BWA-3) (10 mg ml1 in blocking solution) or a rabbit polyclonal anti- and plated on tryptic soy broth agar, and the plates were incubated
acetyl histone H4 antibody (Cell Signaling, Danvers, MA) was for 1 day at 371C.
incubated with the membrane overnight at 41C. BWA-3 reacts with
histones, H2A and H4 (Monestier et al., 1993). After washing the CONFLICT OF INTEREST
membrane three times with 0.1% TTBS, a horseradish peroxidase- The authors state no conflict of interest.
labeled goat anti-mouse polyclonal antibody (1:5,000 in the blocking
solution; DAKO, Carpinteria, CA) or a horseradish peroxidase-
labeled goat anti-rabbit polyclonal antibody was incubated with the REFERENCES
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2496 Journal of Investigative Dermatology (2009), Volume 129

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