Article Type: Review Article

Accepted Article
Doxycycline As An Anti-Inflammatory Agent: Updates in Dermatology.

Running title: review of anti-inflammatory mechanisms of doxycycline with possible

relevance in dermatologic diseases.

Authors and Affiliation:

M. Henehan, B.S., M. Montuno, M.D. and A. De Benedetto, M.D.

Department of Dermatology, College of Medicine, University of Florida, Gainesville (FL -

USA)

All Authors have no conflict of interest to declare. All Authors have read the manuscript and
have approved submission.

Corresponding Author: Anna De Benedetto, M.D.

Assistant Professor of Dermatology

College of Medicine, University of Florida

4037 NW 86 Terrace

Gainesville, FL 32606

Phone: 352-294-4427

adebenedetto@ufl.edu

This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/jdv.14345

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 Abstract

Doxycycline, a tetracycline antibiotic, is widely used in the field of dermatology for its
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antibiotic properties, anti-inflammatory properties and good safety profile.

Over the past decades, numerous studies have clarified some of the anti-inflammatory
mechanisms of doxycycline. In this review article, we aimed to provide an update on recent
data on the anti-inflammatory properties of doxycycline and its potential role in cutaneous
inflammatory diseases. Better understanding of these mechanisms might offer the practicing
clinicians a better use of this therapeutic tool. In addition, research in this field could help
clarify pathogenic aspects of inflammatory dermatologic diseases responsive to this
medication.

Further research is needed to fully elucidate the potential of doxycycline as an anti-

inflammatory agent and the development of new topical vehicles could open ways to new
therapeutic possibilities for dermatologists.

Introduction

Doxycycline is a semisynthetic 2nd generation tetracycline that was chemically

derived from the 1st generation tetracyclines originally discovered in Actinomycetes soil
bacteria1. Chemically, doxycycline differs from tetracycline only by the position of a single
hydroxyl group; yet, it has several unique and useful properties that differentiate it from other
tetracyclines. Doxycycline was FDA approved as an antibiotic in 1967 and to this day
remains in the antibiotic arsenal of most clinicians1. Primarily, doxycycline has been used as
a broad-spectrum bacteriostatic agent that inhibits bacterial protein synthesis by targeting
the 30S ribosomal subunit of both gram-positive and gram-negative bacteria2. It was not
until a 1983 report of minocyclines ability to reduce collagenolytic activity that researchers
began considering the anti-inflammatory effects of tetracycline class drugs3. What has
followed is an explosion of research and reports of doxycyclines therapeutic potential
beyond the scope of merely being an antibiotic.

Group 2 or 2nd generation tetracyclines, which include doxycycline and minocycline,

have been found to be many times more lipophilic than the 1st generation or Group 1
tetracyclines4, allowing for greater distribution to tissues such as the skin. Doxycycline was
demonstrated to have a bioavailability of over 80% when taken orally5 with absorption being
relatively less affected by food intake than other tetracyclines6. Following the recent
advances in knowledge of the cutaneous anti-inflammatory effects of doxycycline, the field of

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 dermatology has begun expanding its use to diseases with a predominantly inflammatory
(rather than microbial) pathophysiology. This review seeks to provide a cohesive overview of
recent data that has emerged in aiding our understanding of doxycyclines influence on
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physiology and its mechanisms of action that are relevant to cutaneous inflammatory
disease states.

Methods & Results

Searches were done utilizing PubMed search engine. Terms used were doxycycline
along with mechanism, target and inflammation. Once a target of doxycycline was identified,
a new search using doxycycline plus the target/pathway name as search terms was
undertaken (see subtitles of results section for each target identified and searched).
Antibiotic resistance data was searched using a combination of the terms doxycycline,
tetracycline, antibiotic, resistance and subantimicrobial dose. Date filter ranged from its year
of FDA approval (1967) to present (2016). Our review focuses on the anti-inflammatory
mechanisms of doxycycline, as its anti-microbial properties have been described in detail
elsewhere2.

Terms used to look at clinical applications of doxycycline were doxycycline,

dermatology and anti-inflammatory. Once a clinical application was identified, new searches
were performed including the name of the disease. Additional reports were gathered from
review of the textbook titled Treatment of Skin Disease: Comprehensive Therapeutic
Strategies7.

Matrix Metalloproteinases (MMPs)

MMPs are key multi-domain zinc-containing endopeptidases involved in several

biological processes. Under homeostatic conditions, MMPs are not detectable (or are very
low) in the skin. However, expression of MMPs increases during wound healing, cancer, as
well as inflammatory conditions. MMPs are the most extensively studied targets of
doxycycline to date. Interestingly, doxycycline in subantimicrobial doses is the only FDA-
approved drug listed explicitly as an MMP-inhibitor, being used as an adjunct therapy in
adult periodontitis (Periostat; CollaGenex Pharmaceuticals Inc., Newtown, PA, USA)8.

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 MMP-2, MMP-3, MMP-8, MMP-9, and MMP-13 have been observed to be inhibited
by doxycycline in the literature in various models (summary in table 1); however, data on
skin cells are still limited.
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Beginning with MMP-2, also known as gelatinase A, early observations revealed that
doxycycline inhibited the secreted levels of MMP-2 in cell culture of a breast cancer cell line
and noncompetitively inhibited its enzymatic activity9. Another study observed that
doxycycline inhibited the enzymatic action of MMP-2 purified from a mesothelioma cell line10.
Later, a different study added that doxycycline also acted to inhibit MMP-2 by reducing its
expression level three-fold in the aortic wall of aortic abdominal aneurysm patients11. Recent
experiments found that doxycycline reduced pathogenic levels of MMP-2 in rats with induced
periodontitis12.

MMP-3 was first shown to be inhibited by doxycycline at the transcriptional level in

human fibroblasts13. Comparably, doxycycline has been found to suppress expression of
MMP-3 induced in a neuronal cell line model15 as well as in a model of hernia repair in rats16.
A more recent trial in abdominal aneurysm patients showed that doxycycline reduced mRNA
of MMP-3 in the aortic wall tissue14.

MMP-8, also known as neutrophil collagenase, has been noted to be inhibited by

doxycycline both through decreased mRNA levels and protein activity inhibition in
endothelial cells17. In addition, multiple studies investigating doxycycline in periodontitis
patients have noted decreased MMP-8 levels in tissue samples18, 19. This drug has also
proven itself capable of lowering MMP-8 levels in tuberculosis patients, helping limit tissue
destruction20. Trials looking at the effect of doxycycline on abdominal aneurysm patients14
and rheumatoid arthritis21, 22 also found suppressed levels of tissue MMP-8. It has been
observed that doxycycline at pharmacologic concentrations in vitro can inhibit human MMP-8
(and MMP-13) while remaining far below the level needed to inhibit mammalian MMP-123.
Finally, one other set of experiments found that doxycycline was unable to inhibit MMP-8
activity when pH is 7.1 or lower24. This last finding is intriguing given the fact that the pH
gradient in the skin has previously been found to range from 4-6, lower than most of the
body25.

MMP-9, also known as gelatinase B, has largely mirrored the inhibition observed with
MMP-8 as the two have commonly been investigated together. In the skin, immune cells
(e.g. mast cells and dendritic cells) as well as keratinocytes produce and secrete MMP-
926, 27
. MMP-9 is involved in cleavage and degradation of several components of the
basement membrane allowing immune cells to transmigrate in the epidermis during the
inflammatory process. In vitro experiments have shown that doxycycline lowers both mRNA

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 levels and enzyme activity of MMP-9 in endothelial cells13 and in a breast cancer cell line9.
Tissue levels of MMP-9 were also reduced in trials of doxycycline in patients with abdominal
aneurysm14, periodontitis28, acute coronary syndrome29, tuberculosis30 as well as
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endometrial levels31. Increased MMP-9 expression and/or activity has proved to be present
in several dermatologic conditions, including autoimmune disease, pemphigoid, rosacea,
atopic dermatitis, and psoriasis32-36.

MMP-13 has been observed to be inhibited by doxycycline both via expression level
modulation in a periodontitis model18 and via inhibition of recombinant human MMP-13
enzymatic activity21. Doxycycline has also been observed to inhibit MMP-13 at both the
mRNA and protein levels in epithelial cells of human eye cornea cells37. Of note, MMP-13
has been found in various epithelial cancers as well as in fibroblasts in chronic cutaneous
ulcers38.

Protease-Activated Receptor 2 (PAR2)

A proposed target of doxycycline is PAR2 (summary in Table 1). This receptor was
first described via molecular cloning and in vitro expression as a transmembrane receptor
with a tethered extracellular amino terminus small peptide, which acts as an activating ligand
to the receptor after being cleaved off 39. PAR2 is expressed in the skin by suprabasal
keratinocytes as well as inflammatory cells40. It can be activated by endogenous serine
proteases and by numerous environmental proteases (e.g. bacteria products, dust mites,
allergens)41. Recently, several studies have shown that PAR2 has involvement in
keratinocyte differentiation as well as in skin hydration, neurogenic inflammation, and
sensation of pruritus40, 42-44. A study using models of toxic and allergic contact dermatitis in
mice and humans found that PAR2 activation induces multiple cutaneous effects including
edema, plasma extravasation, leukocyte recruitment, upregulation of cell adhesion
molecules, and increased pro-inflammatory cytokines45. Additionally, when PAR2 is
stimulated by proteases it has been observed to induce delay of epidermal permeability
barrier recovery46.

Doxycycline appears to inhibit PAR2 activation directly and indirectly. One study
utilizing epidermal keratinocyte culture suggested that doxycycline (as well as tetracycline)
inhibited IL-8 secretion induced by PAR2 agonist peptide SLIGKV-NH247. Further study are
needed to clarify if this effect is due to a direct effect of doxycycline on PAR2 function and/or
expression or instead secondary to the effect of doxycycline on other mediators.
Interestingly, another set of experiments found that doxycycline indirectly inhibited the

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 proteolytic activation of trypsin like serine proteases in the skin, most notably through
preventing the activation of Kallikrein5 (KLK5)48. This indirect inhibition was observed as the
result of doxycycline inhibiting various MMPs, which were responsible for the proteolytic
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activation of KLK5 and other trypsin like serine proteases that contribute to epidermal
inflammation48. Interestingly, other researchers discovered that activated KLK5 can serve as
a proteolytic activator of PAR249.

In all, PAR2 as a target of inhibition is a significant consideration because its

activation leads to increased production of inflammatory cytokines such as IL-1 and TNF-,
provides a stimulus to release more MMPs into the local environment.

Leukocyte Chemotaxis

Another broad category of targets inhibited by doxycycline is in the area of leukocyte

chemotaxis, having been noted in tetracycline as far back as 197850. Follow-up studies
added that doxycycline reduced both random migration and guided chemotaxis of
neutrophils51, 52. More recent studies have begun to elucidate the molecular mechanisms by
which doxycycline has this effect. One mechanism has been the observed reduction in
monocyte chemoattractant protein 1 (MCP-1) in A549 human lung epithelial cells, which
appear to be the result of a doxycycline-induced decrease in rate of MCP-1 mRNA
production53. Other research has shown that systemic doxycycline can reduce neutrophil
chemotaxis into lung airspace of mice54 and into the aortic wall of human aortic abdominal
aneurysm patients14. However, one study highlights a need for more investigation as it found
that doxycycline had an inhibitory effect on neutrophil adherence at low drug concentrations,
while it enhanced binding at higher concentrations55.

Notably, doxycycline may also indirectly inhibit leukocyte chemotaxis. For instance,
PAR2 activation has been found to attract leukocytes in human and mouse contact
dermatitis models45 and has been associated with MCP-1 release56. Additionally, as
mentioned above doxycycline reduces levels of IL-8 (a neutrophil attractant chemokine) in
epidermal keratinocytes as a downstream result of PAR2 inhibition47. In parallel,
doxycyclines indirect inhibition of KLK5 also has a noted downstream effect of decreasing
active cathelicidin in the skin, which also acts as a leukocyte chemoattractant57. It is yet
unclear to what degree impaired leukocyte chemotaxis augments the reduction in tissue
MMP levels noted in the previous section.

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 Influence on Inflammatory Cytokines

Doxycyclines effects on cytokines are complex, but its ability to reduce levels of
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inflammatory cytokines is especially relevant given the emergence of new clinical indications
for this drug. Specifically, there are several publications noting that doxycycline reduces the
pro-inflammatory cytokines IL-158, IL-628, 29 and TNF-28, 59 (in addition to IL-8 discussed
above). In concert with what has been described above, reduction by doxycycline of
inflammatory chemotactic cytokine levels such as IL-8 and MCP-1 are also key aspects of
this effect on inflammatory cytokines. Mechanistically, these effects have been at least
partially documented by findings that doxycycline suppresses activation of the NF-kB
pathway28, 49 and by direct inhibition of TNF--Converting-Enzyme (TACE)60. As described
in the PAR2 section above, the PAR2 pathway is also likely to be involved in pro-
inflammatory cytokine production45.

Nitric Oxide Synthase

Doxycycline has been found to decrease pathogenic Nitric Oxide Synthase (NOS)
levels in an osteoarthritis model, through a mechanism whereby the drug reduces the
stability of NOS mRNA61, 62. A follow up study further noted that doxycycline promoted post-
transcriptional modification of inducible NOS (iNOS), leading to accelerated mRNA
degradation in mouse macrophages63. More recent studies have shown that this translates
to noticeably lower levels of iNOS activity in mice59 as well as lower levels of markers of
nitrosative stress in the serum and gingiva of periodontitis patients64.

IgE Pathway

Recent data has begun to show that doxycycline (and other tetracycline) exerts an
inhibitory influence on the IgE pathway65, 66
. One study found that in vitro, doxycycline
inhibited IgE production mediated by peripheral blood mononuclear cells harvested from
asthmatic patients67. Moreover, other researchers observed that doxycycline was able to
reduce peak IgE levels in vivo and inhibit memory IgE responses in vitro68. Similarly, mice
with experimentally induced allergic conjunctivitis that were treated with doxycycline showed
decreased IgE release from B cells and less histamine release from mast cells that was
hypothesized to be due to modulation of a PI3K/Akt pathway69. Doxycycline (and other
tetracyclines) appears to reduce IgE production by targeting T cell pathways, by suppressing
expression of the phosphorylated MAP Kinase p3865.

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 Radical Scavenging / Antioxidant Effect

An interesting effect of doxycycline (as well as tetracycline) is its apparent ability to

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scavenge radicals and reduce oxidative stress. One of the early published results
demonstrated that doxycycline protected against peroxynitrite-induced degradation of 1-
antiproteinase70. Later investigations found that doxycycline decreased the oxidative stress
index of gingival tissue in rats with periodontitis71. Other experiments showed that
doxycycline showed radical scavenging capabilities in a DPPH assay59 as well as inhibited
the production of superoxide radical in a chemiluminescence model72. Once again, it is yet
uncertain how much the antioxidant effects of doxycycline are due to direct radical
scavenging versus reduced leukocyte migration, as leukocytes produce many reactive
oxygen species.

Other Targets

A few other targets of doxycycline inhibition are worth mentioning despite less
volume of research behind them. First is the observation that doxycycline may be able to
hinder granuloma formation (in vitro model) via inhibition of Protein Kinase C, with one study
finding a dose-dependent relationship in this effect73. Furthermore, there have been some
case reports that minocycline has been effective in treating patients with silicone-induced
granulomas74, 75.

Phospholipase A2 (PLA2) is known for its activity in releasing arachidonic acid from
phospholipids for use in producing eicosanoids that play a key role in inflammatory
processes. Investigators have found that doxycycline is capable of inhibiting PLA2 by
interfering with the Ca2+ binding loop of the enzyme active site, though with a lower affinity
than minocycline76. This lower affinity of doxycycline agrees with the initial report stating that
the inhibitory concentration of doxycycline for PLA2 is considerably higher than that required
to inhibit other targets such as MMPs77.

Apoptosis pathways are also emerging as possible targets. Doxycycline has been
observed to inhibit poly-(ADP-ribose) polymerase-1 (PARP-1), an enzyme that promotes cell
death and inflammation78 as well as promote apoptosis in cancer cells by activating caspase
pathways79.

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 Antibiotic Resistance Concerns

As doxycycline has broad-spectrum antibiotic activity at its classical dose of 100 mg

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twice daily, clinicians have expressed concern about emerging resistance with prolonged
use. Accordingly, many have attempted using doxycycline at a subantimicrobial dose to
selectively utilize its anti-inflammatory properties. Several groups have investigated this
sub-antimicrobial dose and have yet to find evidence that it contributes to resistance80-83.
One group found no evidence that sub-antimicrobial dose doxycycline exerted any effect on
composition or resistance levels of fecal and vaginal microflora80. Likewise, one study found
that sub-antimicrobial dose doxycycline did not cause statistically significant changes in
doxycycline-resistant microbes of the oral microflora81. Similarly, subgingival microflora of
periodontitis patients treated with sub-antimicrobial doxycycline (20 mg twice at day) did not
show bacterial shifts beyond what could be attributed to anti-inflammatory host benefits82.
Interestingly, a study found that Propionibacterium acnes did not exhibit a significant
increase in antibiotic resistance after 6 months of treatment with subantimicrobial dose
doxycycline (20mg, twice daily)83. Given how few studies have undertaken this question of
resistance with use of subantimicrobial dose doxycycline, additional data is needed to
alleviate the concerns about this potential threat.

Discussion and Conclusions

Tetracyclines are the most prescribed oral antibiotics by dermatologists, used

primarily for acne vulgaris and rosacea with a long satisfactory track record of efficacy and
safety84. The promise of doxycyclines anti-inflammatory activity is most evident in the fact
that it has been approved by the FDA for treatment of periodontitis85 and rosacea86 in the
sub-antimicrobial dose. These indications are the first of their kind in recommending the
drug for its inhibition of MMPs function. Growing evidence from clinical trials strongly
supports the anti-inflammatory label of this antibiotic. In a double-blind, randomized,
placebo-controlled study in patients with papulo-pustular rosacea, doxycycline (40 mg/daily,
modified release capsule) induced clinical improvement as it compared to placebo.
Importantly, the treatment with doxycycline was associated to reduced expression of
cathelicidin, KLK5, and MMPs expression and activity. The hypothesis is that by down
regulating MMPs, doxycycline normalized cathelicidin levels in rosacea patients33.

Various inflammatory processes have been implicated in acne vulgaris, and many
receptors and mediators of these pathways are targeted by doxycycline. A study by Lee et
al. showed that PAR2 activity is increased on keratinocytes in acne vulgaris; increased

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 activity further stimulates human beta defensin-2, LL-37, MMP-2, MMP-3, MMP-9, and
MMP-1387. As aforementioned, PAR2 and several MMPs are inhibited by doxycycline, along
with downregulation of the NF-kB pathway. The downregulation of NF-kB is of significance
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as it has been found to be highly activated in acne vulgaris with resultant elevated TNF-
and IL-1 secretion, both of which further amplify NF-kB signaling88. Thus, doxycyclines
several anti-inflammatory effects make it a favorable choice in managing inflammatory acne
vulgaris. Clinical trials are needed to further investigate the effect of doxycycline (or
tetracycline in general) at the subantimicrobial dose for the treatment of acne. Notably, a
few small trials indicate that such a regime was indeed effective, well tolerated, and
importantly no detectable antimicrobial effect on the skin flora was found83, 89.

In addition to rosacea and acne, the anti-inflammatory use of doxycycline has been
suggested for other dermatologic diseases (Table 2), including bullous dermatoses (MMP-2,
MMP-9, neutrophil chemotaxis), cutaneous sarcoidosis (granuloma formation/PKC), kaposis
sarcoma (MMP-2), and neutrophilic dermatoses (neutrophil chemotaxis)90-94.

Interestingly, new investigations have implicated that tetracycline can target PAR2
and NF-kB as well as the IgE pathway, which are relevant in the pathogenesis of atopic
dermatitis66, 95. As minocycline has been shown to decrease T cell proliferation in vitro and
both minocycline and doxycycline have been shown to decrease granuloma formation in
vitro, this observation can provide further credence to the use of doxycycline in sarcoidosis
and granuloma annulare91, 92. Moreover, the anti-collagenase properties of doxycycline have
been cited in its efficacy in treating alpha-1 antitrypsin deficiency96. A summary of anti-
inflammatory clinical uses (and doses) of doxycycline and level of evidence is provided in
Table 2.

Importantly, in the above mentioned dermatologic diseases (with exception of

Rosacea and Periodontitis) doxycycline is used for the anti-inflammatory properties;
however, the doses used (100 mg or more daily) are still anti-microbial. Although much has
been investigated, more research is still needed to fully understand the therapeutic potential
of doxycycline (e.g. anti-inflammatory vs sub-microbial doses) in the field of Dermatology.
Notably, several clinical trials are currently investigating the use of doxycycline (as well as
minocycline) in topical formulations97-99, which may become of greater use in the future of
dermatologic practice. One trial made use of an experimental doxycycline hydrogel on
diabetic foot ulcers, which demonstrated improved lesions and a decrease in local serine
proteases60. Also, related compounds with a similar mechanism of action may prove to be
clinically helpful. Two such related compounds are chemically modified tetracyclines

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 (CMTs), which have had the antimicrobial side group of normal tetracyclines chemically
removed100, and polyenolic zinc-binding MMP-inhibitors (PEZBINs), which are a derivative of
the natural product curcumin101.
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Overall, doxycycline appears to have a myriad of uses beyond its initial discovery as
an antibiotic that are of great use to clinicians in dermatology. Although the medical field has
yet to work out all the details of doxycycline, our hope is that this review serves as a
springboard for both improved clinical use of doxycycline and in research to help further
relieve certain patients inflammatory disease states.

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