Severe Acne in Female Patients Treated with Isotretinoin is associated with Dysbiosis and its Consequences

Severe acne is mainly caused by Propionibacterium acnes (PA) and is sometime treated with isotretinoin. Propionibacterium species may alter intestinal mucosa and isotretinoin may induce mucosal side-effects. We investigated whether severe acne (treated or not with isotretinoin) is associated with dysbiosis and its consequences. Methods: All female patients consulting for the first time the same gastroenterologist were included into a retrospective observational cohort with a large control group in order to evaluate the incidence of adverse events in gastroenterological consultations. Those with a medical history of severe acne -treated with isotretinoin or antibiotic therapy-were compared to a control group. The study stopped when 1,000 patients were included into the control group. Age, Body weight, Body Mass Index, exhaled hydrogen or methylacetate after fasting (a marker of dysbiosis and malabsorption), medical history of allergy or auto-immune disease, oral herpes simplex 1/2 replication (confirmed by qPCR) were collected. Anxiety and depression scores were calculated. Results: 1,054 patients were enrolled: 1,000 control patients; 28 with severe acne not treated with isotretinoin (group 1) and 26 with isotretinoin (group 2). Group 2 (isotretinoin) has a lower body weight (56.9 ± 9.9 versus 61.1 ± 15 kg; p<0.05) and Body Mass Index (20.84 ± 3.8 versus 22.85 ± 5.2; p<0.05) than the control group (despite being younger (40 ± 10 years versus 50 ± 16; p<0.05). Group 2 (isotretinoin) presents with dysbiosis: higher levels of H2 (7.3 ppm ± 7 versus 4.6 ± 6.9; p<0.05) and methylacetate (5.4 ± 2.6 versus 3.9 ± 4.4; p<0.05). It also presents more frequently with oral herpes simplex (30.4% versus 21.4%; p<0.05) or allergy (34.6% versus 15.7%) which suggests dysimmunity. Group 1 (without isotretinoin) does not experience dysbiosis. Anxiety and depression scores were higher in patients with acne (treated or not with isotretinoin). The incidence of isotretinoin-associated events is equal to 2.6% of gastroenterological consultations. Conclusion: In female patients, isotretinoin is associated with dysbiosis, malabsorption and dysimmunity.


Introduction
Small Intestinal Bowel Overgrowth (SIBO) is due to bacterial proliferation in the jejunum.These bacteria are consuming unabsorbed disaccharides because of mucosal enzymatic deficiencies or excessive intakes of sweetened food.Drug-induced mucosal atrophy belongs to Severe acne was defined as an acne resistant to local treatments and which required either antibiotic therapy or isotretinoin for at least 3 months.
Patients were recruited during a consultation in a gastroenterological ward.They were split into three groups: one group with severe acne treated with oral antibiotic therapy more than three months (group 1), one group with severe acne treated with oral isotretinoin (group 2) and one control group.
The study should stop either after one year on recruitment or when 1,000 female patients were included into the control group.This design enables to calculate the incidence of intestinal dysbiosis associated with severe acne treated or not with isotretinoin, in gastroenterological consultations (Table 1).

Patients
Inclusion criteria: All female patients consulting for the first time the same gastroenterologist were included into a prospective observational study.
All patients signed a written consent before the consultation which includes breath test, ultrasound, elastometry and viral detection in saliva.
Exclusion criteria: Male patients were not included because gender may influence small gut absorption, dysbiosis, tobacco and alcohol abuse, immunity and Body Mass Index.Lack of signed consent or missing medical or biological data was an exclusion criteria.

Data collected
Age, Body weight, Body Mass Index, exhaled hydrogen or methylacetate after fasting (measured by the MX6 of Gazdetect France), medical history of allergy or auto-immune disease, oral herpes simplex 1/2 replication (medical history confirmed by qPCR run into a central laboratory; material: Amplix® from Alldiag®; reagents: Bioneer®) were collected.
Propionibacterium acnes (PA) is frequently involved in acne and can be detected by PCR in 82.8% of lesions [28][29][30].Propionibacterium species may adhere to intestinal mucosa [31].They secrete hyaluronidase and can alter intestinal mucosa [32][33][34].Microbiomes may associate several aggressive bacteria such as PA or Acinetobacter species and lead to synergic destructive and atrophic effect on mucosa.
Patients with acne may receive isotretinoin which mucosal sideeffects profile is well documented [35].This mucosal toxicity is also illustrated by the possible occurrence of ulcerative colitis [36][37][38][39].Isotretinoin affects also stem-cells renewal [35,[40][41][42][43]. May an isotretinoin-induced mucosal atrophy occur, a long-term or even a permanent effect could be expected; leading to severe consequences, repeated consultations and explorations in gastroenterology, and therefore to health expenditures.As a matter of fact, Irritable Bowel Syndrome is known to induce important health costs [44][45][46].
Since isotretinoin remains a frequent treatment of acne, its safety profile should be further investigated.We therefore decided to perform a retrospective cohort to investigate whether severe acne treated with isotretinoin or severe acne treated with antibiotic therapy alone is associated with dysbiosis and its consequences.We enrolled a large number of patients in order to get the incidence of isotretinoinassociated malabsorption observed by a gastroenterologist.The inclusion stops as soon as the number of patients enrolled into the control group reaches one thousand.
The incidence of the event should be put into perspective with the one of other drug-induced malabsorption such as olmesartan-related celiac-like disease [47].

Statistics
Comparisons of means were performed using independent samples T tests.Comparisons of percentage used two-sample t-tests.

Results
1,054 patients were enrolled: 1,000 control, 28 patients with severe acne not treated with isotretinoin (group 1) and 26 patients treated with isotretinoin (group 2).Isotretinoin was given for 6 to 12 months at the usual recommended dose: 0.5 to 1 mg/kg.None reported severe toxicity which required discontinuation.70% reported skin or mucosal dryness during or after isotretinoin therapy.The delay between the start of the intake of isotretinoin and the diagnosis of dysbiosis/malabsorption ranges between 18 to 25 years with a median period equal to 22 years.Group 2 has a lower body weight (56.9 ± 9.9 versus 61.1 ± 15 kg; p<0.05) and Body Mass Index (20.84 ± 3.8 versus 22.85 ± 5.2; p<0.05) than the control group (despite being younger (40 ± 10 years versus 50 ± 16; p<0.001).Group 1 presents also with lower body weight than the control group.There was no difference with group 2 as far as body weight and age are concerned.
Group 1 presents neither with dysbiosis, nor with increased rate of oral herpes simplex or allergy.There was no difference between the control group and group 2 as far as depression was concerned.However, when patients with severe acne were pooled (54 patients treated or not with isotretinoin) the depression score was higher in patients with severe acne (5.2 ± 3.6 versus 6.6 ± 4.0; p>0.05) (Table 2).
The incidence of isotretinoin-associated dysbiosis is equal to 2.6%.
HP favours gastric mucosal atrophy [48] without any jejunal involvement [49].Desulfovibrio favours ulcerative colitis [50][51][52][53].However, there is no argument in favour of any Desulfovibrio-induced mucosa alteration of the small gut.Acinetobacter favours mucosal atrophy [25][26][27].Therefore, although PA cannot directly favour colitis, other associated bacteria belonging to the Prevotella-enterotype may induce mucosal inflammation.However, there is no argument for any jejunal adverse effect of PA or of any associated bacteria from the Prevotella-enterotype.
On the contrary, mucosal side-effects are well known side-effects of isotretinoin [35] and ulcerative colitis have been reported with this medication [36][37][38][39].However, jejunal involvement or severe malabsorption has not yet been reported.
According to Melnik BC [35], the unifying mechanism of all isotretinoin-induced adverse effects is the apoptosis of stems cells which involves neural crest cells (explaining teratogenicity), hippocampal neurones (depression), epidermal keratinocytes and mucosa cells (muco-cutaneous side-effects), hair follicle cells (telegenic effluvium), intestinal epithelial cells (inflammatory bowel disease), skeletal muscle cells (myalgia and release of creatine kinase) or hepatocytes (release of transaminases and very low-density lipoproteins).
In addition, isotretinoin normalizes [58,59] exaggerated TLR-2mediated responses in acne patients [60,61].Since TLR2 plays many positive physiological roles, deleterious consequences of its blockade are expected.TLR2 regulates the production of neurotrophic factors in intestinal smooth muscle cells and promotes survival of enteric neurons and glial cells [62,63].TLR2 controls mucosal renewal.Cells kinetics of villous columnar epithelial cells are modulated by TLR2 and controls the proliferation of indigenous bacteria [64][65][66].TLR2 enables local tolerance via Bacteroides fragilis.It discriminates between pathogens and symbiotic bacterial molecules in a process that engenders commensal beneficial colonization [67,68].TLR2 controls indigenous bacteria proliferation in the upper alimentary tract.Lack of TLR-2 contribute to the settlement of undesirable bacteria, especially Gram-positive, or candida [69,66,70,4].Duodenal villous atrophy has be associated with drugs such as Olmesartan [3] or mefenamic acid, [71] and mimics celiac disease with weight and fat loss.The involvement of stem cells apoptosis and mTORC1 blockade has never been reported with any drugs; except isotretinoin.The maximum reported range of incidence of Olmesartan-associated celiac-like disease is 22 cases/10 4 exposed patients [47].The estimated incidence of isotretinoin-induced celiaclike disease is 10 times higher.
Dysbiosis is easily detected by breath tests.The fermentation of sugars produces soluble gas which circulate to the lungs where they are exhaled [72,73].Methylacetate, which belongs to the group of Volatile Organic Compounds, is particularly valuable to distinguish between various types of dysbiosis including small gut inflammation [74].
The increase in exhaled methylacetate is suggestive of dysbiosis associated with the PA-associated enterotype [74].
Acne is associated with mood disturbances, especially anxiety [75] and its treatment alleviates symptoms [76,77].Isotretinoin has previous been implicated in the worsening of depression [78,79].New data do not confirm such a role or even conclude to a positive psychological effect after acne alleviation [80][81][82][83][84][85].According to our results, acne it-self (and therefore PA and the associated enterotype) could explain the difference observed between HAD scores in the control group and in patients with severe acne.
The study only included females.Male patients were excluded.They present with different clinical pictures and deserve a specific publication.The conclusion of this study is therefore limited to female patients.

Conclusions
The study included only female patients.The conclusions are therefore only relevant for females.Isotretinoin is associated with dysbiosis, malabsorption (with visceral fat decrease) and signs of dysimmunity.Symptoms develop more than 20 years after the intake of the medication taken at usual range and duration.A facilitating role of Propionibacterium acnes cannot be excluded.Isotretinoin is known to impair stem cells renewal and TLR2 expression in the mucosa of the small gut.These pharmacological effects, explaining the efficacy of the medication on acne ("the metabolic syndrome of the pilosebaceous gland") may induce the progressive atrophy of the jejunal mucosa and its long-lasting consequences.No therapy is available yet.Patients, prescribers and authorities should be aware of this adverse event which incidence is high (2.6% of outward gastroenterological consultations) especially when multi-annual surveillance for several years are expected.

Figure 1 :
Figure 1: Lipid synthesis in the pilosebaceous follicle is favoured by the western diet (according to Melnik BC et al. [55]) and mTORC1

Table 1 :
Design of the retrospective cohort study of Clinical and Cosmetic Dermatology Open Access Journal from the Hamilton Anxiety Depression scale.Normal level of faecal elastase excluded exocrine pancreatic insufficiency.

Table 2 :
Data collected for the three groups of patients (all females) who consulted in a gastroenterological ward.Retrospective cohort study *P<0.05 between control and acne or between control and isotretinoin, but not between acne and isotretinoin § P<0.05 between acne and isotretinoin or between control and isotretinoin; not between control and acne !P<0.05 between control and isotretinoin only; not between acne and isotretinoin **p>0.05 between control and pooled data from the 54 patients with acne treated or not with isotretinoin