Potential role of microorganisms in thepathogenesis of rosacea

Anna D. Holmes, PhDFort Worth, Texas

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Rosacea is a skin condition of abnormal inflammation and vascular dysfunction. The active contribution ofa microbial agent in the development or progression of rosacea continues to be debated. Research supportsthe presence of commensal Demodex folliculorum mites at increased density in the skin and associatesHelicobacter pylori infection of the gut with rosacea. Fewer studies implicate Staphylococcus epidermidis,Chlamydophila pneumoniae, and the Demodex-associated bacteria Bacillus oleronius. No research,however, provides a mechanism by which colonization by a microorganism translates to manifestationof the condition. Prevailing and emerging principles in the biology of the microbiome and thepathophysiology of rosacea may help to reconcile these lingering questions. Here the microorganismsimplicated in rosacea are reviewed and the reaction of the microbiome to inflammation and to changes inmicroenvironments and macroenvironments are discussed to explain potential roles for microorganisms inrosacea pathophysiology. ( J Am Acad Dermatol 2013;69:1025-32.)

Key words: commensal; Demodex folliculorum; Helicobacter pylori; immunoreactivity; mechanism;microbiome; pathophysiology; rosacea.

Multiple pathways and events that contri-bute to rosacea pathophysiology haverecently been defined,1,2 however, the

presence of a microorganism as a contributingagent remains controversial. Studies have reportedincreased microbial loads or immune hyperreactivityto the following microorganisms in patientswith rosacea: Demodex folliculorum, Bacillusoleronius, Helicobacter pylori, Staphylococcusepidermidis, and Chlamydophila pneumoniae.3-7

As several of the identified microorganisms arecommensal species, it is difficult to prove thattheir presence is associated with disease. Currently,2 general hypotheses explaining the role ofmicroorganisms in rosacea are commonly accepted:(1)microbes are causative agents; or (2)microbes areinnocent bystanders.

To better understand the microbiology of rosacea,we must evaluate not only the microorganismsthemselves but also the microenvironments andmacroenvironments in which they reside. Here wediscuss physiologic and inflammatory influences onthe rosacea microbiome, review the existing

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ing provided by Galderma Laboratories LP, Fort Worth, TX.

osure: Dr Holmes is employed by Galderma Laboratories LP,

rt Worth, TX.

int requests: Anna D. Holmes, PhD, Galderma Laboratories LP,

501 North Freeway, Fort Worth, TX 76177. E-mail: anna.

lmes@galderma.com.

literature on the microbes of rosacea, and proposean alternative theoretical framework for understand-ing the complex role of microorganisms in thedevelopment or progression of rosacea.

LESSONS FROM THE MICROBIOMEThe physiology of the skin

Human skin is composed of dozens of distinctiveand diverse microenvironments for colonization bymicroorganisms. Multiple factors influence microbialcolonization, including aerobicity, humidity, tem-perature, pH, lipid composition, age, and sex.8-10

Endogenous factors such as sweat, sebum, andhormone production can differ greatly betweenindividuals, as can exogenous influences such asclimate, diet, toiletries, and medications.10-13

The microbes inhabiting a given microenviron-ment are diversified based on the suitability of theseconditions for growth of each individual species. Theresident microflora of moist occluded areas, forexample, varies greatly from that of exposed areaswith high sebaceous gland density.14

Published online September 9, 2013.

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� 2013 by the American Academy of Dermatology, Inc.

http://dx.doi.org/10.1016/j.jaad.2013.08.006

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During normal homeostasis, the microbes inha-biting a microenvironment reach a balance. If afactor affecting the growth or survival of amicroorganism changes, a dysbiotic shift may occur.In rosacea, multiple factors are altered comparedwith unaffected facial skin, including temperature,fatty acid composition, connective tissue, and

CAPSULE SUMMARY

d Several microorganisms have beenshown to be increased orimmunoreactive in patients with rosacea.

d An understanding of the microbiomeand rosacea pathophysiology mayexplain alterations in and reactivity tomicrobial populations.

d Awareness of disease mechanisms areimportant for targeted patient care and

vascular structures.5,15-17

Rosacea also typically mani-fests in individuals overthe age of 30 years, and cer-tain symptoms are moreprevalent depending onsex. Taken together, the mi-croenvironment of rosacea-affected skin may besubstantially altered, result-ing in a shift in the propor-tion or speciation of theresident microflora.

selection of therapeutics.

The immune system andthe microbe

Immune surveillance is crucial for clearanceof unwanted microorganisms and tolerance ofcommensal microbial species. Pattern recognitionreceptors expressed in the skin initiate a host ofantimicrobial activities upon contact with pathogen-associated motifs.18,19 Commensal microorganismssuch as S epidermidis provide a second layer ofdefense by producing antimicrobial factors thatinhibit growth of pathogens such as S aureus, whilesparing other commensal species.20-23

The immune system tolerates normal flora underhomeostatic conditions; however, it may becomeactivated during or contribute to dysbiotic episodes.In dermatologic conditions such as psoriasisand dandruff, a shift in the resident microbiotaaccompanies disease.24,25 In atopic dermatitis,disease flares are associated with an increase inboth pathogenic and commensal staphylococcalspecies in lesional skin.26 Misdirected antimicrobialactivities likely contribute to observed microbialshifts.27-29

In rosacea, expression of pattern recognitionreceptors TLR2, NALP3, and the antimicrobialpeptide cathelicidin is increased in multiple cutane-ous subtypes.30-33 Furthermore, macrophages, mastcells, and T-helper 1 cells are present at higherdensities than in controls.2 These cells andmediators may increase sensitivity to or eliminationof pathogenic or commensal microbes. As with skinphysiology, the inflammatory state of skin caninfluence composition of the resident microbiota.

The influence of systems biologyCross-talk between the skin and other organ

systems can be readily assumed based on thenumber of skin conditions that commonly comani-fest with noncutaneous disorders.34 It has recentlybeen postulated that a specific communicationaxis may exist between the skin, the gut, and the

brain, as both gut and skinexhibit similar neuronal andinflammatory activity.35-39

The first conclusive evidencewas reported in a mousemodel where oral probio-tics improved stress-inducedcutaneous neurogenic in-flammation.37 Intriguingly,augmenting the gut micro-flora was also central to thisset of experiments.

A growing body of workhas also emerged recogni-zing a reciprocal relationshipbetween microorganisms

and the macroenvironment beyond which theyimmediately reside. This concept is exemplifiedby rheumatoid arthritis studies where altered gutmicroflora directly induce joint inflammation.40,41

Broader examples include the hygiene andmicroflora hypotheses, which suggest increasedcleanliness and antibiotic use, respectively, disruptimmunologic tolerance to normal flora,42-45 possiblyexplaining increasing atopy and irritable bowelsyndrome in developed countries. These illustrationssupport the idea that intricate communication axesmay exist between the microbiome and multiple,distal sites within an individual.

Although theoretical, these philosophies can beextrapolated to rosacea by a striking number ofparallels. Gastrointestinal comorbidities,46,47 neuro-genic inflammatory pathways and mediators,2,33 andmicrobial irregularities of both skin and gut haverepeatedly been reported across rosacea subtypes.Furthermore, oral antibiotics commonly used for thetreatment of rosacea can result in drastic shifts inresident microflora of skin and gut and may disruptimmune tolerance. Based on these findings, it ispossible that themicrobiome in patients with rosaceais disrupted locally or distally and subsequentlyinfluences disease manifestation or progression.

MICROBES ASSOCIATED WITH ROSACEADemodex folliculorum

D folliculorum is a species of commensalsaprophytic mite that colonizes pilosebaceous folli-cles of human skin. The prevalence of Demodex

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steadily increases with age to nearly 100% in lateadulthood.48-50 Because of its universal presence,Demodex infestation, or demodicosis, is typicallydefined as Demodex counts of 5 or more mites/cm2

and is readily measurable by standardized surfaceskin biopsy specimen or exudation of sebaceousgland contents.51-53

The clinical symptoms of demodicosis includeerythema, telangiectases, itching, and scaling49,54

and can manifest as rosacea-, perioral-, orgranulomatous-like dermatitis or blepharitis.53,55-57

Likewise, elevated Demodex loads have beenreported in rosacea, seborrheic dermatitis, perioraldermatitis, and blepharitis,58-61 which resembledemodicosis clinically.62-64 A genetic predispositionfor demodicosis has been reported65,66; however,explanations for a pathogenic mechanism remaintheoretical.4,66-70

Considerable research has examined Demodexdensity in patients with rosacea. A previous reviewsummarizing controlled studies from 1993 through2001 revealed numerically and, where applicable,statistically significantly higher Demodex density onthe cheeks of patients with papulopustular rosacea(PPR), and average Demodex densities exceeding5mites/cm2.49 Additional recent reports demonstratea 5.7-fold increase in Demodex density acrosserythematotelangiectatic rosacea (ETR) and PPR32

and significantly higher Demodex infestation rates,typically ranging from 35% to 50%56,71-73 but as highas 90% in 1 PPR study.74

The effect of different treatment modalities onconcomitant reduction of Demodex density androsacea symptoms has also been evaluated.75-78

Both parameters consistently improved, althoughthere was no continuity in treatments administeredacross studies. The largest investigation reportedthat significantly higher reduction of Demodex didnot translate to better efficacy in a head-to-headtreatment comparison in patients with PPR.75

Amechanism forDemodex in rosacea has recentlybeen proposed,32 based on previous findings thatdust mite allergens are capable of activatingNOD-like receptors in vitro.79 NALP3, increased4-fold in ETR and 10-fold in patients with PPR, isdirectly involved in activation of the interleukin-1b inflammasome and may explain activation of theinnate immune response in rosacea.32

Bacillus oleroniusB oleronius are nonmotile gram-negative endo-

spore forming bacteria not currently recognized ascommensal organisms of human skin. First describedin 1995 after isolation from the hindgut of a termite,80

B oleronius has since been cultured from 1 Demodex

mite isolated from a patient with rosacea4 and severalpatients with blepharitis.81 The pathogenic potentialof B oleronius is currently unknown.

In 2007, proteins isolated from B oleroniuswere found to trigger a proliferative response inperipheral blood mononuclear cells of 73% ofpatients with PPR but only 29% of control subjects.4

Similar statistically significant serum immunore-activity has since been reported in ETR and ocularrosacea.82,83 Innate immune mediators induced byB oleronius have not been measured in patients withrosacea; however, neutrophils isolated from healthyvolunteers and exposed to B oleronius proteinsexhibited increased migration and production ofmatrix metalloproteinase-9, interleukin-8, andtumor necrosis factor,84 which mirrors inflammatoryactivation in patients with PPR.30,31,33

Staphylococcus epidermidisS epidermidis are the most prevalent commensal

bacteria of healthy human skin. These gram-positivebacteria have long been regarded as symbiotic flora,as their presence has been associatedwith protectionof the skin from pathogenic species includingS aureus.20,21,23 Pathogenic behavior of S epidermi-dis is typically limited to invasion of an alreadycompromised skin barrier, such as chronic wounds85

and internally inserted medical devices.86

Investigations into the prevalence and growthcharacteristics of S epidermidis in patients withrosacea have indicated these bacteria are singularlyabundant in pustular lesions5,87 and exhibit analtered secretory profile. S epidermidis culturedfrom pustules of 4 patients with rosacea produceddifferent proteins at increased rates when incubatedat increased temperatures. S epidermidis isolatesfrom rosacea pustules were also beta-hemolytic,which may indicate secretion of virulence factorsnot present in control isolates.5,88

Helicobacter pyloriH pylori, helical gram-negative bacteria that reside

in the stomach, are one of the most common humanpathogens, likely infecting more than 50% of thegeneral population.89,90 Although the majorityof individuals are asymptomatic, H pylori arerecognized as a causative factor of chronicgastritis, peptic ulcers, and gastric cancers.91 H pyloriseropositivity has also been linked with cardiovas-cular, respiratory, neurologic, and autoimmunedisease, as well as rosacea, psoriasis, and idiopathicurticaria in the skin.92-95

H pylori produce numerous cytotoxins thatcontribute to inflammation in the gastric mucosa.91,93

As a result, a defined host response is mounted that

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includes production of cytokines96 and infiltration ofmonocytes, neutrophils, and lymphocytes.91-93

Since the original publication of a possibleassociation between H pylori and rosacea in 1994,6

a wealth of controversial literature has bothconfirmed96-101 and refuted97,102-104 this hypothesis.Similarly, studies evaluating the efficacy of H pylorieradication therapy in rosacea have met withconflicting results.98,99,102,104,105 Because antibiotictherapy is the standard of care for both PPR andH pylori infection, some believe results may beconfounded. This is exemplified by reports oftemporary improvement in rosacea during eradica-tion treatment that were not sustained after treatmentcompletion97 and of greater improvements in thePPR subtype.104 In 2 independent studies, only afterseparating the results of patients who had not usedantibiotics was a strong correlation found betweenH pylori infection and rosacea.106,107

Research focused specifically on the H pylorivirulence factor cytotoxin-associated gene A(CagA) protein also appears to circumvent conflict-ing reports. Results from multiple rosacea subtypesestablished that 67% of patients harbor H pyloristrains positive for CagA,98 and 75% havemeasurableCagA-reactive antibodies.99 These results correlatedwell with patients who experienced both rosaceaand gastric symptoms,104 allowing the authors toconclude a possible association with CagA1 H pylorigastritis, rather than simply H pylori infection.

Mechanisms of H pylori-induced rosacea havebeen proposed, which include triggering ofinflammation by H pylori cytotoxins and gastrin-induced flushing107; however, substantial evidencethat rosacea symptoms occur in response to H pyloridoes not currently exist.

Chlamydophila pneumoniaeThe bacteria C pneumoniae are obligate intracel-

lular pathogens and a common causative agent ofrespiratory diseases such as pneumonia and asthma.A possible link to atherosclerosis and central nervoussystem disorders has also been identified.108

In a single uncontrolled study of 10 patientswith rosacea, C pneumoniae antigen was detectablein 40% of malar skin biopsy specimens, andC pneumoniaeereactive antibodies were present in80% of serum samples.7

DISCUSSIONMechanisms explaining how alterations in micro-

bial populations or immunoreactivity contribute torosacea pathology have not been defined. There is aconstant interplay among the skin, microorganisms,and the environment that maintains a delicate

balance, and disturbance of this balance caninfluence both the microflora and skin health.Building on this microbiome principle, understand-ing the possible role of the identified microbialspecies in the initiation or progression of rosaceabecomes more tangible and also more complex thanpreviously imagined, as proposed in Fig 1. How eachspecies may contribute to the proposedmodel basedon the research reviewed here is discussed below.

Steps 1 and 2Immune surveillance is conducted in part by

pattern recognition receptors that bind pathogen-associated ligands, leading to enhanced immuneactivation. In rosacea, TLR2 up-regulation has beenconfirmed in the PPR subtype, and NALP3 isup-regulated in both PPR and ETR. Althoughcommensal microbes do not activate these receptorsunder normal circumstances, receptor hyperre-activity or decreased tolerance could explainheightened sensitivity and triggering of inflamma-tory pathways by commensal or noncommensalrosacea-associated agents. Such activities may leadto the initial clinical manifestation of rosacea.

Step 3Changes in skin physiology can immediately

impact microbial growth, metabolism, and specia-tion. In rosacea skin, alterations in temperature,lipids, and dermal structures may provide asuitable physiologic environment for increasedDemodex growth in PPR and ETR, alteredS epidermidis secretions in rosacea pustules, andthe presence of C pneumoniae antigens.

As with physiologic change, inflammation canalso accompany shifts in microbial populations.Increased cathelicidin expression and leukocyteactivity have been described in all cutaneoussubtypes of rosacea, and each can effectivelyeliminate susceptible, targeted bacteria. After suchmicrobicidal episodes, opportunistic growth ofunaffectedmicroorganisms into the space previouslyoccupied by the eliminated organism can occur,which may explain increased Demodex loads inrosacea. Similarly, synergistic production ofantimicrobial factors by S epidermidis and neutro-phils in rosacea pustules may explain the absence ofadditional species in these lesions.

Step 4Alterations in microflora may impact innate

immune responses by initiating or exacerbatingcellular activities described in steps 1 and 3, furthercontributing to inflammation or the development ofpapulopustular or granulomatous lesions.

Fig 1. Proposed role for microorganisms in rosacea. Step 1: Unaffected rosacea-prone skin hasincreased expression of microbe sensing machinery and normal flora. Step 2: Commensalagents may act as trigger factors for rosacea inflammation through activation of patternrecognition receptors (PRR) or altered tolerance. Step 3: Physiologic and inflammatory changesin rosacea skin may create a microenvironment that is suitable for altered growth or metabolismof microbial species. Step 4: Alterations in commensal flora or introduction of noncommensalagents may result in further activation of inflammatory pathways, worsening of disease, orappearance of subtype-specific symptoms. Step 5: An immune response that results inclearance of the exacerbating agent may impart temporary relief from rosacea symptomsand a return to microbial homeostasis. AMP, Antimicrobial peptide.

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Unlike innate immunity, the adaptive immuneresponse in rosacea remains largely undefined.Heightened antibody reactivity to noncommensalB oleronius, H pylori, and C pneumoniae has beenreported; however, B-cell activation has not beendescribed, and increased B-cell density has onlybeen documented in phymatous forms of disease.2

Alternately, a CD41 T-cell infiltrate has beenrepeatedly described in all cutaneous subtypes ofrosacea both in the presence and absence of anidentified microorganism.2,72,74 In spite of thesediscrepancies, an adaptive immune responseprovides another theoretical mechanism foraccelerated inflammation following alteration ofthe resident microflora.

Increasing evidence supports the hypothesis of acommunication axis linking the skin and the gut.Although H pylori infection is limited to the

upper gastrointestinal tract, similar patterns ofcellular activation and inflammatory cytokine pro-duction, as well as gastrin-induced flushing, mayexplain cutaneous manifestation or exacerbation ofinflammation in ETR and PPR by an unknownpathway.

Step 5An effective immune response may temporarily

control microorganisms and influence the cyclicnature of rosacea.

The hypotheses presented here are reflective of aconcentration of research in rosacea pathophysio-logy and the microbiome of the skin. With theexception of Demodex and H pylori, the associationof rosacea and microorganisms is supported bylimited, small, and underpowered studies.

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DECEMBER 20131030 Holmes

Not all patients with rosacea exhibit changes in orimmunoreactivity to the microbes examined here,and eradication therapy is not universally effective.Moreover, disease flares are commonly associatedwith known nonmicrobial triggers (eg, ultravioletexposure, exercise, alcohol) and sustained, atleast in part, by nonimmune-related mechanisms(eg, neuropeptides).

Areas of future research prioritization to discernthe role of microorganisms in rosacea pathophysiol-ogy include independent examination of the rosaceamicrobiome associated with disease progression,subtypes, and the gastrointestinal tract. Additionalexploration of genetic associations and biomarkersto predict cellular heterogeneity, activation status,clinical presentation, and impact on microorganismsis also necessary.

CONCLUSIONSThe information presented here suggest that

although microorganisms may not be central causa-tive factors in rosacea pathogenesis, they are likelyaltered in multiple subtypes of rosacea and may actas trigger factors or potentiators of inflammation inan undefined subset of predisposed patients.

The author would like to recognize MatthewMeckfessel, PhD, for assistance in figure development.

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