Irritable Eye Syndrome: Neuroimmune Mechanisms and Benefits of Selected Nutrients

Accepted Manuscript

Irritable Eye Syndrome: Neuroimmune Mechanisms and Benefits of SelectedNutrients

Janos Feher, MD, PhD Erika Pinter, MD, ScD Illés Kovács, MD, PhD ZsuzsannaHelyes, MD, ScD Agnes Kemény, PhD Adrienn Markovics, MD, PhD Rocco Plateroti,MD Aloisa Librando, MD Filippo Cruciani, MD

PII: S1542-0124(13)00166-3

DOI: 10.1016/j.jtos.2013.09.002

Reference: JTOS 79

To appear in: Ocular Surface

Received Date: 21 March 2013

Revised Date: 21 July 2013

Accepted Date: 9 September 2013

Please cite this article as: Feher J, Pinter E, Kovács I, Helyes Z, Kemény A, Markovics A, Plateroti R,Librando A, Cruciani F, Irritable Eye Syndrome: Neuroimmune Mechanisms and Benefits of SelectedNutrients, Ocular Surface (2014), doi: 10.1016/j.jtos.2013.09.002.

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http://dx.doi.org/10.1016/j.jtos.2013.09.002

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ACCEPTED MANUSCRIPTIrritable eye Sydrome 1 Feher

SECTION: Original Research Report, Gary N. Foulks, MD, Editor

TITLE: Irritable Eye Syndrome: Neuroimmune Mechanisms and Benefits of Selected Nutrients

AUTHORS: Janos Feher, MD, PhD,1,4 Erika Pinter, MD, ScD,2,5 Illés Kovács, MD, PhD,3

Zsuzsanna Helyes, MD, ScD,2,5 Agnes Kemény, PhD,2 Adrienn Markovics, MD, PhD,2 Rocco

Plateroti, MD,1 Aloisa Librando, MD,1 and Filippo Cruciani, MD1

SHORT TITLE: IRRITABLE EYE SYNDROME/Feher et al

FOOTNOTES

Accepted for publication November 2013.

From the 1Ophthalmic Neuroscience Program, Department of Visual Science, Sapienza University

of Rome, Italy, 2Department of Pharmacology and Pharmacotherapy, University of Pecs, Hungary, 3Department of Ophthalmology, Semmelweis University, Budapest, Hungary, 4Nutripharma

Hungaria Ltd, Budapest, Hungary, and 5PharmInVivo Ltd, Pecs, Hungary.

This work was supported by Uj Széchenyi Terv Fund of the Hungarian Government, a KMOP-

1.1.4-11/B-2011-0024 to the Nutripharma Hungaria Kft, Budapest, Hungary.

Dr. Feher holds patents related to the composition of Framelim®.

This paper was presented in part at the ARVO Annual Meeting, May 6-10, 2012, Fort Lauderdale,

FL, USA

Single-copy reprint requests to Janos Feher, MD, PhD (address below).

Corresponding author: Janos Feher, MD, PhD, Via Sardegna, 139, 00187 Rome, Italy. Tel/fax: +39-

06- 42010510. Email: [email protected].

ABSTRACT Previous studies showed comorbidity of some ocular, enteral, and affective symptoms

comprising irritable eye syndrome. Aims of the present study were to learn more about the

pathogenic mechanisms of this syndrome and to evaluate benefits of food supplements on these

disorders. In in vitro assay, Lactobacillus acidophilus lysate inhibited interleukin (IL)-1β and tumor

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necrosis factor (TNF)-α generation of lipopolysaccharide (LPS)-stimulated macrophages in dose-

and size-dependent manner. For a prospective, open-label phase I/II controlled clinical trial, 40

subjects affected by ocular dysesthesia and hyperesthesia and comorbid enteral and anxiety-

depression symptoms were randomly assigned either into the treated group, which received a

composition containing probiotic lysate, vitamins A, B, D and omega 3 fatty acids, or into the

control group, which received vitamins and omega 3 fatty acids. For reference, 20 age- and sex-

matched healthy subjects were also selected. White blood count (WBC) and lymphocyte and

monocyte counts, as well as IL-6 and TNF-α levels, were significantly above the reference levels in

both treated and control groups. After 8 weeks, WBC and lymphocyte and monocyte counts, and

cytokine levels significantly decreased, and ocular, enteral, and anxiety-depression symptoms

significantly improved in the treated group as compared to the control group. This proof-of-concept

study suggested that subclinical inflammation may be a common mechanism connecting ocular,

enteral, and anxiety/depression symptoms, and supplements affecting dysbiosis may be a new

approach to treating this syndrome.

KEY WORDS anxiety, cod liver oil, constipation, depression, diarrhea, incontinence, interleukin

(IL)-6, monocyte count, ocular dysesthesia, ocular hyperesthesia, probiotic lysate, TNF-α, vitamin

B, white blood cell count

Outline

I. Introduction

II. Microbiota-Brain Interactions

III. Influences of Probiotics on Neuroimmune Regulation

IV. A Proof of Concept Study on the Benefits of Nutrition Support to Irritable Eye

Syndrome

A. Methodology

1. In Vitro Study Design

2. Patients and Treatment

3. Procedures

4. Statistical Analysis

B. Results

1. In Vitro Anti-inflammatory Effects of Lactobacillus Lysate

2. Anti-inflammatory Effects of Probiotic Lysate, Cod Liver Oil, and B

Vitamins

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3. Changes in Quality of Life

V. Discussion

A. Mechanism of Disease

B. Mechanism of Nutritional Benefits

C. Ocular Symptoms

D. Enteral Symptoms

E. Anxiety and Depression Symptoms

VI. Summary and Conclusions

I. INTRODUCTION

Clinical studies in the early 1990s found for the first time that ocular hypersensitivity to

environmental influences and enteral disorders associated with certain food intake may have

comorbidity with anxiety-depression symptoms. This triad of symptoms was termed irritable eye

syndrome (IES), and dysfunction of neurohormonal and immune systems was thought to be the

underlying pathogenic mechanism.1 Further studies confirmed the comorbidity of ocular and enteral

symptoms in inflammatory bowel diseases2,3 and in functional gastrointestinal disorders,4 as well as

the comorbidity of ocular and psychiatric symptoms in depression syndrome,5,6 in depression of the

elderly population,7 and in depression associated with post-concussion syndrome.8 Recent studies

have revealed some new details of bidirectional communications between gut and brain that may

contribute to better understanding of the pathophysiology of these evident clinical comorbidities.9

II. MICROBIOTA - BRAIN INTERACTIONS

It is generally accepted that a vast bacterial community (up to 100 trillion bacteria) called

microflora or microbiota lives in symbiosis with the host enteral tract, and gives the major input for

the bidirectional communication between the enteral neurohormonal-immune system and the brain

neurohormonal-immune system, called the microbiota-gut-brain axis.10 The afferent arc of this

communication in which bacterial products or fragments from the microbiota influence brain

functions may act: 1) through stimulating sensory fibers of vagal nerve or spinal cord, which

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transfer information to specific brain centers and release neuropeptides (substance P, CGRP,

somatostatin), and 2) through the bloodstream by release of gut hormones such as 5-

hydroxytryptamine (5-HT) from enteroendocrine cells, of cytokines from mucosal immune cells,

and of bacterial molecules (fatty acids, GABA, 5-HT precursors) to the brain.

The efferent arc of this communication is formed by parasympathetic fibers of the vagal nerve

and sympathetic fibers from sympathetic ganglia. They influence the autonomous motility and

secretory functions of the intestine and exert anti-inflammatory and/or proinflammatory effects on

gut macrophages. Emotions, stress, and trauma to the brain can also affect gut functions and the

microbial composition through the release of stress hormones or sympathetic neurotransmitters.11

Several life-style factors may cause the perturbation of this microbiota-host symbiosis, called

dysbiosis, which is characterized by deterioration of the epithelial functions and subsequent

impairment of digestion, absorption, and utilization of nutrients, as well as by enhanced

translocation of bacterial products or fragments and the subsequent upregulation of the enteral

neurohormonal-immune system followed by upregulation of the brain neurohormonal-immune

system.12 Very similar, but less extensive, communication has been described between the ocular

surface and the adequate brain centers, called the microbiota-eye-brain axis. The neural circuit is

formed by afferent sensory fibers of the trigeminal nerve and the efferent parasympathetic fibers of

the facial nerve.13 Conjunctiva-associated lymphoid tissue represents the immune component of this

ocular neuroimmune system.

Recently, a DNA sequencing-based survey of the bacterial population at the conjunctiva has

revealed an unexpectedly diverse microbial community containing commensal, environmental, and

opportunistic pathogenic bacteria.14 All these findings justify an assumption that the triad of ocular,

enteral, and affective symptoms may refer to microbiota-host dysbiosis in the gut and on the ocular

surface. Thus dysbiosis may be a new target for treating this syndrome.

III. INFLUENCES OF PROBIOTOCS ON NEUROIMMUNE REGULATION

Probiotic bacteria, a subgroup of microbiota, have been widely used to treat enteral dysbiosis

and related symptoms since ancient times. Animal studies showed that in addition to their local

effects, probiotics administered orally also alleviated the anxiety-like behavior in a colitis model15

and depression symptoms in a myocardial infarction model.16 These effects were enhanced by a

combination of probiotics and omega 3 fatty acids.17 Clinical experience confirmed that probiotics

may have benefits on symptoms of depression syndrome18 and brain injury.19 Interestingly, another

line of research revealed that both local and systemic effects may also be evoked by lysate of

probiotics rather than live ones, called probiotic paradox20 ; killed probiotics showed greater effects

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on chronic diarrhea,21 on irritable bowel syndrome,22 and in stimulating innate immunity than did

their live forms.23 However, benefits of probiotic lysate on ocular and affective disorders have not

yet been studied.

IV. A PROOF OF CONCEPT STUDY ON THE BENEFITS OF SELECTED

NUTRIENTS IN THE IRRITABLE EYE SYNDROME

This current single-center, prospective, open-label, phase I/II clinical trial investigates the safety

and potential efficacy of a food supplement that contains fragmented lysate of probiotics, cod liver

oil (vitamin A, Vitamin D, and omega 3 fatty acids), and B vitamins. The selection of probiotic

strains was based on previous studies, which showed their efficacy in restoring gut functions.24

Addition of cod liver oil and B vitamins was justified by epidemiologic data showing lower levels

of essential nutrients such as vitamin A and D and omega 3 fatty acids, as well as some of the B

vitamins in neuropsychiatric diseases, and by the well-known role of these ingredients in the

physiology of the neuroimmune system.25 The primary endpoint of this study was to show that

probiotic lysate in combination with A,B, D vitamins and omega 3 fatty acids ameliorate at least

one marker of systemic inflammation, while a secondary endpoint was to demonstrate improvement

in at least one of the ocular, or enteral or psychiatric symptoms, as compared to the control.

A. Methodology.

1. In Vitro Study Design

Inflammatory cells were obtained from the peritoneal cavity of male CR1/CD mice 4 h after

i.p. lipopolysaccharide (LPS) injection (300 µl per animal in 300-µg/ml solution, Salmonella

enterica lipopolysaccharide, Sigma-Aldrich, St. Louis, USA). Thirty-six mice were used for the six

experimental groups. The abdominal cavity was washed by 2.5-ml cell culture medium (RPMI

1640, Sigma, St. Louis, MO, USA) supplemented with 10% fetal calf serum under sterile

conditions. The lavage fluid was collected in ice-cold tubes to obtain peritoneal macrophages for

the cultures. Then 100-µl samples of the lavage fluid were injected into 800-µl culture medium in a

24-well plate. In the first step, dose-dependent effects (1, 10, and 100µg/ml) of heat-killed

Lactobacillus acidophilus ATTC 4356 lysate containing 109 CFU/g (Harmonium International Inc,

Mirabel QC Canada) were tested, and for further studies 100 µg/ml LBL were used. For

determining the size-dependent effects, LBL were exposed to ultrasonication using S220 High

Performance Ultrasonicator (Covaris, Inc, Woburn, Ma.USA) to reach either 5-micron or 1-micron

particle size. The cell cultures were incubated as shown in Table 1.

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The plates were then incubated for 8 h (in the case of interleukin [IL]-1β) or 32 h (in the case of

tumor necrosis factor [TNF]-α) in CO2-incubator at 37°C. The contents of the cells were then

collected and centrifuged for 5 min at 12.500 rpm. The concentration of the inflammatory cytokine

IL-1β and TNF-α was measured from the supernatants by sandwich ELISA. IL-1β BD OptEIA

ELISA set (cat. Nr. 559603; BD Biosciences Eastern Europe, Heidelberg, Germany) and TNF-α

DuoSet ELISA (cat. Nr. DY410; R&D Systems, USA), respectively.

2. Patients and Treatment

For human studies, 20 affected subjects (age: 45.15±4.73 y) were randomly assigned to the

treated group and 20 affected subjects (age: 45.95±6.65 y) were randomly assigned to the control

group. The inclusion criteria for IES were the comorbidity of the ocular, enteral and affective

symptoms as evaluated by questionnaire (see details in Section IVA.3: Procedures).

Exclusion criteria were:

1) Abnormal tear secretion and/or tear film stability (for example, established dry eye

syndrome [DES]), Sjögren and non-Sjögren autoimmune keratoconjunctivitis sicca, previous injury

or permanent damage to the eyeball from any cause, hormonal replacement therapy, ongoing

allergic or infective disease of the eye or adnexes).

2) Inflammatory bowel diseases (Crohn disease, ulcerative colitis, diverticulitis),

contemporary use of antibiotics, steroid or nonsteroidal anti-inflammatory treatment or any chronic

treatment potentially affecting intestinal mucosa.

3) Established anxiety or depression or any neuropsychiatric disease under treatment.

4) Advanced systemic and organ diseases.

Affected subjects in the treatment group received three softgels per day, each containing

lysate of Lactobacillus acidophilus ATCC 4356 (1.25x109 CFU) and Bifidobacterium longum

ATCC 15707 (1.35x109 CFU), vitamin A (0.15 mg), vitamin D (1.08 mcg) and EPA (44 mg), DEA

(58 mg) fatty acids in form of cod liver oil, and a mixture of vitamins B1 (thiamin 0.5 mg), B3

(niacin 5 mg), B6 (piridoxin 0.5 mg), B9 (folat 0.075 mg) and B12 (cyanocobalamin 0.5 mcg) (brand

name: Framelim®, Nutripharma Hungaria, Ltd, Budapest, Hungary). Affected subjects in the

control group received supplements containing cod liver oil and the same B vitamins. Dosage of

each compound complied with the recommended dietary allowance (RDA). The eating and

treatment regimens were the same for both groups. These studies were conducted in early spring

time and lasted for 8 weeks. In addition, 20 age- and sex- matched subjects comprised a healthy

group, which was used to establish reference values for laboratory tests and for clinical symptoms.

3. Procedures

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At the time of inclusion into the study, a simplified questionnaire was used to evaluate the

quality of life related to the specific symptoms in both healthy and diseased groups (Tables 2 and

3). This questionnaire comprised three sets of questions and evaluated the frequency of symptoms

using scores as follows: never=0; sometimes=1; frequently=2; always=3.

Routine laboratory blood count testing was performed to measure absolute white blood cell

(WBC), lymphocyte (Ly) and monocyte (Mo) counts by flow-cytometry, and IL-6 and TNF-α

levels were measured by ELISA, as described for in vitro studies. After 8 weeks of treatment both

questionnaire and laboratory tests were repeated for treated and control patients.

4. Statistical Analysis

For in vitro studies, the Mann-Whitney nonparametric test was performed. In all cases a p value

<.05 was considered to be significant. For human studies, statistical analyses were performed using

Statistica 6.0 (Statsoft Inc., Tulsa, OK); in all analyses a p value <.05 was considered statistically

significant. Normal distribution assumption could be accepted for all parameters according to the

Shapiro-Wilks W-test. The Student’s t-test for unpaired samples was used for group comparisons

and the Student’s t-test for paired samples was used for the analysis of repeated measures in the

study group.

The local medical ethics review board approved this study (University of Pecs, Hungary),

animal studies were conducted in accordance of ARVO Statement for the Use of Animals in

Ophthalmic and Vision Research, and human studies were in accordance with the spirit of the

Declaration of Helsinki.

A. Results

1. In Vitro Anti-inflammatory Effects of Lactobacillus Lysate

First, the dose-dependent effects of Lactobacilus lysate (LBL) on generation of IL-1β and

TNF-α of monocytes with or without LPS challenge was tested in vitro. LBL alone in doses of 1,

10, and 100 µg/ml enhanced proinflammatory cytokine production, but inhibited the LPS-induced

IL-1β and TNF-α release from peritoneal macrophages of mice in a dose-dependent manner. The

effect of LBL in 100 µg/ml concentration proved to be significant in this model (data not shown).

Second, we tested the particle size-dependent effects of LBL on generation of IL-1β and TNF-α of

peritoneal macrophages with or without LPS challenge and compared the effect of 1-micron (1000

nm) and 5-micron (5000 nm) size LBL in 100 µg/ml concentration. LPS (1 µg/ml) evoked

significant increase of IL-1β concentration in the supernatant of the cell culture after an 8 h

incubation period (586.00±76.45 pg/ml [100%]). LBL in 100 µg/ml dose has shown a significant

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inhibitory effect on LPS-induced IL-1β release from peritoneal macrophages of mice either in 5-

micron size (360.76±24.45 pg/ml [61.4%]) or 1-micron size particles (52.54±19.77 pg/ml [8.1%]).

The latter was approximately 8 times more effective.

LBL (100 µg/ml) treatment itself (290.8±36.61 pg/ml) did not elevate IL-1β concentration

significantly compared to the negative, PBS-saline control (158.08±35.07 pg/ml Figure 1A). LPS

evoked a remarkable increase of TNF-α concentration in the supernatant of the cell culture after a

32 h incubation period (29.43±3.18 pg/ml). LBL in 100 µg/ml dose formulated in both 5-micron

(5000 nm) and 1-micron (1000 nm) particles inhibited the LPS-induced TNF-α release from

peritoneal macrophages, but only the latter one significantly (9.06±0.25 pg/ml). LBL (100 µg/ml)

treatment itself either in 5-micron size particles (12.63±1.69 pg/ml) or 1-micron particles formulas

(9.23±1.47 pg/ml) slightly elevated the TNF-α concentration compared to the PBS-saline control

(Figure 1B).

2. Anti-inflammatory Effects of Probiotic Lysate, Cod Liver Oil, and B Vitamins

Peripheral absolute WBC count was 5681±657, lymphocyte count was 2202±322, and

monocyte count was 341±65 in the healthy group. These values were significantly lower than those

of the diseased-treated and diseased-control groups at the baseline (P<.001), and all these values

remained in the normal range during the follow-up. After 8 weeks of nutritional support, WBC

count significantly decreased in the treated group (from 7489±1861 to 6445±1202; P<.001) and

slightly, not significantly, decreased in the control group (7378±1093 vs 7046±1030; P>.04). At the

same time, monocyte count decreased from 612±89 to 509±99 in the treated group and from

599±70 to 571±72 in the control group. The decrease in monocyte count was significant in the

treated group (P<.001) but not in the control group (P>.05 [Figure 2A and B]). Lymphocyte count

also decreased significantly in the treated group (from 2705±596 to 2217±442; P=.02), but the

decrease in the control group was not significant (2647±546 vs 2505±621; P>.05 [not shown]).

Proinflammatory cytokine IL-6 level was 4.07±1.48 pg/ml and TNF-α level was 7.74±2.18

pg/ml in the healthy group. These values were significantly lower than those of the diseased-treated

and diseased-control groups at the baseline (P<.001 for both parameters). After 8 weeks of

nutritional support IL-6 level significantly decreased from 17.42±10.52 pg/ml to 12.87±7.37 pg/ml

in the treated group (P<.001), while it remained unchanged in the control group (17.21±5.26 vs

17.31±4.96 pg/ml; P>.05). At the same time, TNF-α level decreased significantly from 177.5±114

pg/ml to 131.3±80.9 pg/ml in the treated group (P<.001), but decreased only slightly in the control

group (174.4±81.71 pg/ml vs 165.2±83.67 pg/ml; P>.05 [Figures 2C and D]).

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3. Changes in Quality of Life

Ocular dysesthesia: The mean score in the healthy group was 3.05±1.14, which was

significantly lower than in either of the diseased groups (P<.001) the baseline. After 8 weeks, the

mean score decreased significantly in the treated group (from 9.85±1.39 to 6.65±1.79; P<.001) and

in the control group (9.35±1.35 vs 8.80±1.57; P=.02 [Figure 3A]).

Ocular hyperesthesia: The mean score in the healthy group was 2.3±1.08, which was

significantly lower than either of the diseased groups at baseline (P<.001). After 8 weeks, the mean

score decreased significantly in the treated group (from 9.85±1.57 to 6.8±0.89; P<.001), and in the

control group (9.75±1.57 vs 9.1±0.97; P=.02 [Figure 3B]).

Stool frequency: Abnormal rate of the stool frequency was reported in 4 cases of the healthy

group, in 10 cases of the treated group (<0.05), and in 9 cases of the control group at the baseline.

After 8 weeks, the stool frequency rate was out of the normal in 3 cases of the treated group and in

8 cases in the control group, equivalent with 70% (<0.05) and 12% improvement, respectively.

Stool consistency: Abnormal stool consistency was reported in 4 cases of the healthy group,

in 12 cases of the treated group (<0.05), and in 13 cases of the control group (<0.05) at baseline.

After 8 weeks, the stool consistency remained out of the normal in 6 cases of the treated group and

in 11 cases of the control group, equivalent with 50% (<0.05) and 15% of improvement,

respectively.

Fecal incontinence was not observed in the healthy group, but it was reported in 4 of the 20

cases in the diseased-treated group at baseline. In all cases, it disappeared after 8 weeks of treatment

(<0.05). In the diseased-control group, incontinence was reported in 3 patients at baseline and

remained unchanged after 8 weeks (Table 4).

Anxiety: At baseline, the anxiety subscale in the healthy group was 4.3±1.08, and it was

significantly lower than in the diseased groups (P<.001). After 8 weeks, the mean score decreased

significantly in the treated group from 11.1±2.4 to 8.1±2.07 (P<.001) but not in the control group

(from 11.5±2.14 to 10.4±1.6; P>.05 [Figure 4A]).

Depression: At baseline, the depression subscale in the healthy group was 3.7+1.3, and it

was significantly lower than in the diseased groups (P<.001). After 8 weeks, the mean score

significantly decreased in the treated group (from 10.2±1.44 to 7.8±1.67; P<0.01) and remained

unchanged in the control group (10.4±1.79 vs 10.0±1.52; P>.05 [Figure 4B]).

Framelim® was well tolerated and no adverse effects were detected in this phase I/II pilot study.

V. DISCUSSION

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A. Mechanism of Disease

Our observations demonstrated that the triad of ocular, enteral, and affective disorders may be

associated with systemic chronic subclinical inflammation. We found higher WBC and lymphocyte

and monocyte counts in the study group as compared to the age- and sex-matched healthy group.

Although values were in the normal range for both groups, the difference was statistically

significant for WBC, monocytes, and lymphocytes. Our results of increased WBC count and

lymphocyte count are novel and should be confirmed by further studies in a larger number of cases.

However, the data for monocyte count are in accordance with previous clinical experience, which

showed some correlation between monocyte count and disease activity in inflammatory bowel

disease,26 and in chronic constipation, a cardinal complaint in neurodegenerative diseases.27

Interestingly, in depression the monocyte count was associated with the disease state: increased in

non-melancholic, decreased in melancholic patients.28 In a follow-up study of patients affected by

major depressive disorder, significant increases in the monocyte count were detected, which

decreased with clinical improvement but remained unchanged in treatment-non-responder cases.29

Peripheral blood monocyte count was also a very simple but useful marker for clinical follow-up in

post-concussion syndrome.30

In addition to the increase in monocyte count, we found significantly higher IL-6 and TNF-α

levels in the diseased group as compared to the reference values of the healthy group. Higher

proinflammatory cytokine levels were also observed in enteral diseases.31,32 Previous studies also

reported altered blood cytokine profile of patients affected by depression syndrome33 and in

traumatic brain injury.34 One of the main impacts of our results is that substantial increase in

monocyte count and in proinflammatory cytokine levels may be suitable markers for detecting IES.

B. Mechanism of Nutritional Benefits

Probiotic lysate showed anti-inflammatory effects in our studies. First, in an in vitro model

Lactobacillus acidophilus lysate inhibited IL-1β and TNF-α generation of LPS-stimulated

macrophages in a dose-dependent manner. Furthermore, the inhibition was significantly higher

when smaller particles were used as compared to the inhibitory effects of larger particles,

suggesting that fragmentation may improve the bioavailability of killed probiotics. Previous studies

have shown that both live and heat-killed probiotics can modify release of proinflammatory

cytokines of macrophages: they increase proinflammatory cytokine release of nonstimulated

macrophages, but in contrast, they exerted inhibitory effects on LPS-induced proinflammatory

cytokine release in vitro and in vivo in a dose-dependent manner.35

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Second, our human study confirmed these anti-inflammatory effects. After 8 weeks

administration of a supplement containing Lactobacillus acidophilus lysate and Bifidobacterium

longum lysate, cod liver oil, and B vitamins, we found a significant decrease in WBC, leukocyte

and monocyte counts, and in both IL-6 and TNF-α levels in the study group as compared to the

control group receiving only cod liver oil and B vitamins. Decrease in monocyte count after

probiotic administration was also found by a randomized, double-blinded, placebo-controlled

human clinical trial carried out in healthy adults.36

The orthodox concept on the mode of action of probiotics has supposed that live bacteria

prevent adherence and colonization of pathogens on the host. The current concept suggests at least

three main pathways related to the microbiota-host symbiosis. First, probiotics interact with other

commensal bacteria of microbiota, resulting in a balance between species of bacterial community,

which interfere with the colonization of pathogens and contribute to digestion of some nutrients.

Second, probiotics regulate epithelial functions (epithelial barrier, secretion of mucus and

antibacterial proteins). Third, probiotics perform physiological stimulation of the host

neurohormonal-immune system and metabolism.37 Accumulating evidence suggests that while live

probiotics are needed for the first function, for the second and third functions, killed probiotic

bacteria exert receptor-mediated actions on both mucosal epithelia and dendritic/macrophage cells.

Fragments containing endotoxin bind to the TLR2/4 and stimulate epithelial barrier functions, while

fragments containing probiotic DNA bind to TLR7/9 and stimulate synthesis of cytokines, mucins,

heat-schock proteins, and defensins by epithelial cells.38 Similarly, fragments of probiotic bacteria

bind to both TLR2/4 and TLR7/9 of dendritic cells or macrophages and stimulate innate immunity,

resulting in reduced generation of reactive oxygen species (ROS) and preventing excessive release

of proinflammatory cytokines.39 Furthermore, genomic DNA of probiotics may be responsible for

inhibition of the NFkappaB signaling pathway40 and suppression of TLR2/4 and TLR7/9.39

It should be emphasized that in humans the lysozyme and antibacterial proteins residing in the

enteral mucus serves to kill and fragment Lactobacilli and Bifidobacteria. However, in some

conditions, such as inflammatory diseases, the compromised host epithelia may not be able to

release enough enzymes to kill probiotics and to obtain the necessary active components from the

probiotics. In such conditions, administration of live probiotics may have uncertain or poor benefit,

but probiotic lysate has at least three advantages. The first is higher bioavailability for developing,

maintaining and restoring microbiota-host symbiosis and stimulating immunity.20 Randomized,

double-blind, placebo controlled clinical studies have shown efficacy of orally administred

probiotics lysate on improving immunity. In a multicenter, randomized, controlled trial

Lactobacillus acidophilus lysate showed more efficacy in patients with chronic diarrhea as

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compared to its live counterpart.21 Probiotic lysate stimulated cellular and humoral as well as

mucosal immunity41 augmented acquired immunity of healthy adults,42 increased immunoprotection

of elderly patients,23,43 improved acute diarrhea of children,44 and prevented necrotizing

enterocolitis of neonates.45 Our observations suggested that the IES should be added to the list of

inflammatory diseases in which probiotic lysate demonstrated improvement of innate immunity.

Second, there is no risk of sepsis when probiotic lysate is administered. Several clinical studies

reported cases of sepsis caused by probiotics in patients with compromised intestinal barrier in

inflammatory bowel diseases or impaired immune system (diabetes, major surgery, organ

transplantation, HIV).46

Finally, storage of lysate is safer as compared to storage of live probiotics.

Addition of cod liver oil may enhance the efficacy of probiotics, as it contains Vitamins A and

D, as well as omega 3 fatty acids. Vitamin A increases mucus secretion,47 Vitamin D enhances

mucosal immunity,48 and omega 3 fatty acids in addition to their well-known anti-inflammatory

properties, decrease proinflammatory cytokine production of monocytes49 and improve epithelial

barrier functions.50 Finally, addition of B vitamins to the formulation improves cellular metabolism

and prevents excessive generation of ROS and subsequent neurodegeneration.51

C. Ocular Symptoms

Ocular dysesthesia and hyperesthesia are cardinal symptoms of IES. After treatment, significant

decrease of both symptoms occurred, indicating improvement of trigeminal sensory nerve

functions. It is generally accepted that ocular surface discomfort may come from the tear film

abnormalities known as keratoconjunctivitis sicca or dry eye disease (DED) or dry eye syndrome,52

but growing evidence has suggested that neurohormonal and immune processes may also be

involved in the pathogenic mechanism of this common disease.13,53,54 Although, some overlap

between symptoms of IES and DED is evident, significant differences should be emphasized: tear

flow and stability are normal or increased in IES, while both are altered in DED. Aging, female

preponderance, and hormonal contribution to DED are well documented, while in IES they may

play a minor role. Furthermore, tear substitutes show benefits in DED, while they have very weak

or no effects on symptoms of IES. The mechanism of disease may also be different: in DED

downregulation, while in IES upregulation of ONIS seems to be the main contributor.

D. Enteral Symptoms

In our study the most evident changes were observed in enteral functions: constipation as shown

by stool frequency and consistency significantly improved after 8 weeks of nutritional support. It is

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well known that constipation is frequently encountered in the aging population due to neuronal

losses in both the myenteric and submucosal plexuses of the intestines. These losses start in

adulthood, increase over the rest of the life span, and are specific to cholinergic neurons. Evidence

supports the concept that both the enteral and brain neuroimmune system are more affected leading

to colonic dysmotility in neuropsychiatric diseases, in comparison to aging.55 In these diseases, the

same histopathologic alterations have been described in both enteric and central nervous system.56

Earlier human biopsy studies indicated that inflammation causes structural and functional

alterations to the enteric nervous system. These include changes to the excitability and synaptic

properties of enteric neurons and of the brain. Recent studies showed that the neurodegeneration

also affects the enteric glia, which is associated with mitochondrial abnormalities suggesting that

mitochondrial dysfunction may be an important factor for neurodegeneration in the enteral nervous

system and contributes to gastrointestinal symptoms.57 In addition, we observed improvement also

in fecal incontinence of affected persons, and this was one of the main contributors to improvement

in the quality of life. Similar observation was reported on improvement of fecal incontinence after

one month administration of Lactobacillus lysate in a large number cohort study.22

E. Anxiety and Depression Symptoms

Our studies showed self-reported improvement in anxiety and depression in the study group,

and these findings confirmed previous hypothesis on the presumed effects of probiotics and selected

micronutrients in psychiatric symptoms.58 Multiple molecular mechanisms may contribute to the

beneficial psychological effects of oral probiotics. At least in part, it may come from their anti-

inflammatory effects. In animal models, gut infection caused anxiety-like behavior which was

associated with increased levels of proinflammatory cytokines. Furthermore, cytokine receptors

were found in vagal nerve endings, and antidepressant drugs may have anti-inflammatory effects.

Neurogenic inflammation may also be a contributor, as altered gut microbiota induce higher

substance P release from activated sensory nerves associated with anxiety, depression and

aggression. Administration of probiotics may attenuate these effects.18 Probiotics may also

modulate brain centers for better processing stressful conditions through increased production of

neurotransmitters acetylcholine and GABA by certain bacteria.

Finally, probiotics may modulate glucose metabolism. Recent findings strongly suggest that

mitochondrial dysfunction and subsequent excessive generation of ROS are crucial contributors to

neuroinflammation and neurodegeneration.59 Thus, metabolic effects of this combination of

probiotics, cod liver oil and B vitamins may also be responsible for clinical benefits. Recently, a

clinical trial published by Tillischet al found benefits of probiotics on the activity of brain regions

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that control central processing of sensation and emotion. Consumption for 4 weeks of fermented

milk with probiotics resulted in significant improvement in somato-sensory, viscero-sensory and

affective functions of the brain, specifically in intrinsic activity of resting brain and in task-related

response as shown by functional MRI.60 These observations perfectly fit in with our clinical

findings about the improvement of the oculo-sensory, enteral, and affective functions. Thus the

same probiotic benefits on the neuroimmune system were documented by two different methods.

However, there are some important differences between the two studies. Tillisch et al selected

healthy women without enteral and psychiatric diseases for the study and administered live

probiotics.60 In contrast, at the beginning of our study all subject reported both enteral and affective

disorders, and we administered probiotic lysate with cod liver oil and B vitamins. Now it is well

established that lactic acid bacteria show anti-inflammatory effects. Their genomic DNA was

identified as one of the anti-inflammatory components, and TLR9 is one of the major pathways

responsible for the anti-inflammatory effect of genomic DNA.61 Recent animal and human studies

also suggested that the gut’s response to bacterial DNA may depend not only on the specific type of

bacterial DNA, but also on the host, in particular on the composition of microbiota and the basal

gene expression.62 These provide further evidence of the diversity of probiotic influence on healthy

or diseased enteral mucosa, and this should be considered for developing an adequate probiotic

formulation.

V. SUMMARY AND CONCLUSIONS

This critical review of research findings and our observations support a putative pathogenic

mechanism of IES which comprises the following steps. 1) Various environmental influences,

mainly life-style factors, cause disorders of the intestinal ecosystem (dysbiosis) resulting in

impairment in digestion and absorption of essential nutrients, abnormalities of the epithelial barrier,

and translocation of microbial and food particles. 2) Subsequently, chronic subclinical inflammation

may develop, which may upregulate the neuroimmune system through neuronal and humoral

pathways. 3) Upregulation of the neuroimmune system causes increased sensitivity of the ocular

surface, gut, and brain to environmental challenges and develops the triad of IES: irritable eye,

irritable bowel, and irritable mind.

Common ocular symptoms of dysesthesia and hyperesthesia may call attention to early systemic

neuroimmune dysfunctions related to enteral and neuropsychiatric disorders or diseases. This is one

of several new subspeciality areas in which experts in ocular surface disorders may contribute to

improve quality of life of affected persons. However, evaluation, monitoring, and treatment of these

symptoms need interdisciplinary approach and preparation.

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Results of this proof-of-concept study indicate safety and a potential effect of special nutrients

for the treatment of IES reaching both endpoints. Benefits of a nutritional support on inflammation

and clinical symptoms were significantly higher when probiotic lysate was added to vitamins A, B,

D and omega 3 fatty acids, suggesting that the enteral dysbiosis may be a new target for treating this

syndrome. Furthermore, our in vitro studies showed that probiotic nanoparticles have more benefits

than probiotic particles not of nanoscale. These original findings may be the result of a particular

biological behavior of nanosize particles, and this needs further study.

ACKNOWLEDGEMENT:

Authors express their gratitude to Akos Feher for revising English text.

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LEGENDS FOR FIGURES

Figure 1. Lactobacillus acidophilus lysate inhibit (A) IL-1 beta and (B) TNF-alpha release of LPS

stimulated macrophages in a size-dependent manner in vitro. Columns represent Mean+SEM of IL-

1 β and TNF-α concentrations. Data of LBL-treated groups were compared to the positive and

negative controls. Note: +++: P<.005 vs PBS+saline-treated group, ***: P<.005 vs LPS+saline-

treated group.

Figure 2. Changes in proinflammatory markers (A) absolute WBC count, (B) absolute monocyte

count), (C) IL-6 levels, and (D) TNF-alpha levels after treatment either with probiotic lysate, cod

liver oil and vitamins B (diseased – treated) or with cod liver oil and B vitamins (diseased-control).

Each of these values were significantly elevated in the diseased groups at the baseline compared to

the healthy group (*). After 8 weeks, WBC and monocyte count and IL-6 and TNF-alpha levels

decreased significantly compared to baseline in the treated group, but not in the control group.

Note: ***: P<.001 compared to healthy group; ###: P<.001 compared to baseline in the same

group.

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Figure 3: Changes in ocular symptoms (A) dysesthesia and (B) hyperesthesia after treatment either

with probiotic lysate, cod liver oil and B vitamins (diseased – treated) or with cod liver oil and B

vitamins (diseased-control). Both of these scores were significantly higher in the diseased groups

compared to healthy group at baseline. After 8 weeks these scores decreased significantly both in

the treated group and in the control group. Note: ***: P<.001 compared to healthy group; ###:

P<.001 and #: P<.05 compared to baseline in the same group.

Figure 4: Changes in psychiatric symptoms (A) anxiety and (B) depression after treatment either

with probiotic lysate, cod liver oil and B vitamins (diseased–treated) or with cod liver oil and B

vitamins (diseased-control). Both of these scores were significantly higher in the diseased groups

compared to the healthy group at baseline. After 8 weeks these scores decreased significantly in the

treated group but not in the control group. Note: ***: P<.001 compared to healthy group; ###:

P<.001 compared to baseline in the same group.

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Table 1. Cell culture protocol

Group Mouse cell LBL challenge LPS challenge No. of

mice

1 Peritoneal macrophage LBL (100 µg/ml) of particle size 5000 nm PBS (6 mice)

2 Peritoneal macrophage LBL (100 µg/ml) of particle size 5000 nm LPS 1µg/ml (6 mice)

3 Peritoneal macrophage LBL (100 µg/ml) of particle size 1000 nm PBS (6 mice)

4 Peritoneal macrophage LBL (100 µg/ml) of particle size 1000 nm LPS 1µg/ml (6 mice)

5 Peritoneal macrophage Saline PBS (6 mice)

6 Peritoneal macrophage LBL 100 µg/ml PBS (6 mice)

Abbreviations: LBL: lactobacillus lysate; LPS: lipopolysaccharide, PBS: Phosphate-buffered saline.

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Table 2. Irritable Eye Syndrome Testing Questionnaire for Diagnosis and Treatment Efficacy

Do you feel these symptoms? Never

0

Rarely

1

Often

2

Always

3

Ocular symptoms

- Symptoms of Dysesthesia

Sensation of foreign body or grittiness (sand)

Sensation of dryness

Sensation of scratchiness

Sensation of burning

Sensation of tiredness or discomfort (“I feel my eyes”)

- Symptoms of Hyperesthesia

Sensitivity to wind/air conditioning/ dusts/ smog/ eye-drops

Soreness (mild pain)

Frequent blinking

Episode of excessive tearing

Light-sensitivity/photophobia

The maximum total score for normal was 7 for either dysesthesia or hyperesthesia. Affective symptoms

- Symptoms of Anxiety

I am more irritable than usual.

I get a sort of frightened feeling as if something bad is about to

happen.

Worrying thoughts go through my mind.

I feel anxious when I go out of the house on my own.

I get palpitations or sensation of “butterflies” in my stomach.

I am restless and have to be on the move.

I have panic feeling for apparently no reason at all.

- Symptoms of Depression

I wake early and sleep badly for the rest of the night.

I have a good appetite. (The scoring is reversed.)

I feel miserable and sad.

I feel as if I am slowed down.

I have lost interest in my appearance.

I feel life is not worth living.

I enjoy the things I used to enjoy. (The scoring is reversed.)

The maximum total score for normal was 8 either for anxiety or depression.

Enteral symptoms Number Notes

Stool frequency/week

Items 5 to 7 per week were considered normal.

Events of incontinence/week

None event was considered normal.

Stool consistency by Bristol Stool Chart during the least week

Types 1 and 2 indicate constipation, Types 3 and 4 are the easiest to pass, and was considered normal,

Types 5 and 6 may indicate diarrhea, Type 7 may be a sign of illness, for example food poisoning.

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References

Feher J. Diagnostic value of tear film abnormalities in a new syndrome affecting the neuroendocrine and

immune systems. Adv Exp Med Biol. 1998;438:839-43

Zigmond AS, Snaith RP. The hospital anxiety and depression scale. Acta Psychiatr Scand. 1983

Jun;67(6):361-70.

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Table 4. Changes in the enteral symptoms

Treated group Control group

Healthy Baseline 8 weeks Difference Baseline 8 weeks Difference

Stool frequencies+ 4 10* 3# 7 (70%) 9 8 1 (12%)

Stool consistencies+ 4 12* 6 6 (50%) 13* 11 2(15%)

Fecal incontinence+ 0 4* 0# 4(100%) 3 3 0

+Number of cases out of the normal range. Note: *: p<0.05 compared to healthy, Chi-square test; #: p<0.05

compared to baseline, McNemar Chi-square test for repeated measures.

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Tabl. 3. Bristol Stool Chart

Reference:

Heaton, K W & Lewis, S J Stool form scale as a useful guide to intestinal transit time. Scandinavian Journal

of Gastroenterology, 1997;32:920 - 924.

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Irritable Eye Syndrome: Neuroimmune Mechanisms and Benefits of Selected Nutrients

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