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ORIGINAL ARTICLE
Year : 2022  |  Volume : 35  |  Issue : 2  |  Page : 543-547

Gut lactobacilli profile among a cohort of Egyptian patients with systemic lupus erythematosus


1 Department of Internal Medicine, Faculty of Medicine, Alexandria University, Alexandria, Egypt
2 Department of Medical Microbiology and Immunology, Faculty of Medicine, Alexandria University, Alexandria, Egypt

Date of Submission12-Feb-2022
Date of Decision10-Mar-2022
Date of Acceptance13-Mar-2022
Date of Web Publication27-Jul-2022

Correspondence Address:
Ahmed Shaaban
Alazareeta, Medical Campus, Faculty of Medicine, Alexandria University, Alexandria 21131
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/mmj.mmj_55_22

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  Abstract 


Objective
To evaluate the changes in Lactobacilli and Lactobacillus reuteri among a cohort of Egyptian patients with systemic lupus erythematosus (SLE) and how these changes affect the state of SLE disease activity.
Background
SLE is a heterogeneous chronic autoimmune disease that affects multiple organs with a variety of clinical manifestations. Gut microbiota dysbiosis has been linked to the development or progression of autoimmune disorders.
Patients and methods
This cross-sectional study was conducted on 40 patients with SLE and 30 healthy adults as a control group. Laboratory investigations included antinuclear antibodies, anti-double-stranded DNA, and complement C3 and C4. SLE disease activity index (SLEDAI-2k) was evaluated. Real-time PCR was applied for quantitative assessment of the lactobacillus genus.
Results
The 40 patients comprised 85% females and 15% males, with a mean age of 28.5 ± 7.78 years and disease duration of 11.68 ± 6.8 months. Patients with SLE showed a significant decrease in the abundance of Lactobacillus compared with healthy controls (P < 0.001), and also L. reuteri showed a significant decrease in patients with SLE (P = 0.030). An inverse but nonsignificant correlation between SLEDAI-2K scores for disease activity and Lactobacillus (r=−0.198; P = 0.220) was detected.
Conclusion
There was a significant decrease in the abundance of Lactobacilli and L. reuteri in patients with SLE, so supplementation of Lactobacilli might constitute a future therapeutic target. In addition, an inverse but nonsignificant correlation with SLEDAI-2k was found.

Keywords: gut dysbiosis, Lactobacillus, SLE disease activity index., systemic lupus erythematosus


How to cite this article:
Shaaban A, Alzawawy A, Mahmoud S, Hassan E, Sabry M. Gut lactobacilli profile among a cohort of Egyptian patients with systemic lupus erythematosus. Menoufia Med J 2022;35:543-7

How to cite this URL:
Shaaban A, Alzawawy A, Mahmoud S, Hassan E, Sabry M. Gut lactobacilli profile among a cohort of Egyptian patients with systemic lupus erythematosus. Menoufia Med J [serial online] 2022 [cited 2024 Mar 29];35:543-7. Available from: http://www.mmj.eg.net/text.asp?2022/35/2/543/352205




  Introduction Top


Systemic lupus erythematosus (SLE) is a heterogeneous chronic autoimmune disease that affects multiple organs and tissues and has a range of clinical manifestations ranging from minor disease to a life-threatening course[1]. This is usually the result of autoantibodies and widespread immune complex-mediated tissue damage[2].

The etiology of SLE remains challenging, as there is no single causative agent but an interplay between multiple factors, including genetic, hormonal, and environmental triggers together with autoimmunity [3–5].

The gut microbiota homeostasis offers many benefits that contribute to the individual's health through a variety of biological events such as shaping the intestinal epithelium, improving gut integrity, energy production, synthesis of essential vitamins, protection against pathogens, and modulation of host immunity[6],[7].

Gut dysbiosis which is an imbalance in the gut microbial community between beneficial and pathogenic commensals recently has been found to play a role in autoimmune conditions like rheumatoid arthritis, ankylosing spondylitis, diabetes, inflammatory bowel disease, and multiple sclerosis[8]. Dysbiosis has been linked to the development or progression of autoimmune disorders in several ways, including molecular mimicry, T-cell activation, and excessive production of inflammatory cytokines[9].

Lactobacillus is a genus of gram-positive, rod-shaped, non-spore-forming bacteria that belongs to the Firmicutes phylum. Lactobacillus species constitute a significant component of the human microbiota at a number of body sites, and they are commonly used as probiotics owing to their anti-inflammatory properties[10].

The aim of the work was to evaluate the changes in Lactobacilli and Lactobacillus reuteri among a cohort of Egyptian patients with SLE and how these changes affected the state of SLE disease activity.


  Patients and methods Top


This cross-sectional study was carried out on 40 patients with SLE diagnosed according to the 2012 Systemic Lupus International Collaborating Clinics (SLICC) criteria[11] and 30 age-matched and sex-matched healthy participants as the control group. The patients were recruited from the Rheumatology Unit, Internal Medicine Department, Faculty of Medicine, Alexandria University hospitals, Egypt. The study was conducted in accordance with the ethical guidelines of the 1975 Declaration of Helsinki after it had been approved by the institutional ethics committee, and informed written consent was obtained from each participant.

Exclusion criteria were patients with current or history of gastrointestinal malignancy or diseases, recent surgical intervention of the intestines within the last 6 months, infectious diarrhea within the last 3 months, history of prolonged use of antibiotics within the last month, pregnancy, liver diseases, and patients with other autoimmune rheumatic diseases.

All patients with SLE underwent detailed history taking, systemic physical examination, and assessment of disease activity using the systemic lupus disease activity score (SLEDAI-2k)[12]. Patients were classified according to SLEDAI-2K score into three categories: inactive (SLEDAI: 1–4), mild-moderate activity (SLEDAI: 5–9), and high activity (SLEDAI ≥ 10).

The following laboratory investigations were performed: complete blood picture, erythrocyte sedimentation rate, blood urea, serum creatinine, protein: creatinine ratio in urine, complete urine analysis, antinuclear antibody, anti-double stranded-DNA antibodies (anti-ds DNA), complement 3 (C3), and complement 4 (C4).

Stool samples from patients with SLE and controls were collected for gut Lactobacilli microbiome analysis and then delivered to the Main Microbiology Research Laboratory and stored at − 80°C until further processing. DNA was extracted from 180–220 mg stool samples using the QIAamp Fast DNA Stool Mini Kit (Qiagen, Hilden, Germany) according to the manufacturers' instructions. DNA extracts were stored at − 80°C until PCR testing.

Quantitative real-time SYBR green PCR was applied for identification and quantitation of Lactobacillus and L. reuteri. Oligonucleotide primers targeted at the 16 S ribosomal RNA gene sequences of Lactobacillus and L. reuteri were used. Primers were also used to amplify a conserved 16 S rDNA sequence present in all bacteria (universal primer set, recognizing domain bacteria), the amplification of which served as the denominator against which the amplification of the other bacteria was compared. All the primer sequences were obtained from the previously published studies [13–15]. Primers were commercially obtained (Invitrogen, Carlsbad, CA, USA).

Amplification was performed in a light cycler (Rotor Gene Q; Qiagen, Hilden, Germany) using a SensiFAST TM SYBR No-ROX PCR kit (Bioline Co., London, United Kingdom). In short, forward and reverse primers (4 pmol each) were used in 20-μl reactions containing 2 μl of the DNA extract. PCR amplification was performed with initial denaturation at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s.

Melting curve analysis was performed from 40 to 95°C with a plate reading step after every 1°C and held at a particular temperature for 10 s to check the specificity of the product formed [13–15]. Quantitation of specific bacterial DNA was not expressed as an absolute number but expressed relative to the total (universal) bacterial DNA present in a stool sample by the RQ software (Qiagen).

Statistical analysis of the data

The IBM SPSS software package version 20.0 was used to analyze the data fed into the computer (IBM Corp., Armonk, New York, USA). Numbers and percentages were used to describe qualitative data. The Kolmogorov–Smirnov test was used to verify the normality of the distribution. The range (minimum and maximum), mean, SD, median, and interquartile range were used to describe quantitative data. The significance of the obtained results was judged at the 5% level.


  Results Top


The 40 patients with SLE had a mean age of 32.5 ± 11.5 years and disease duration of 11.68 ± 6.8 months, with 34 (85%) females and six (15%) males. The most common clinical manifestations of patients with SLE in the current study were mucocutaneous, constitutional, and arthritis [Table 1]. The 30 controls had 22 (73.4%) females and eight (26.6%) males with a mean age of 29.4 ± 8.7 years.
Table 1: Distribution of the studied cases according to demographic and clinical manifestation of patients with systemic lupus erythematosus

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Regarding SLE disease activity, eight patients had inactive disease, 19 patients had mild-moderate disease activity, and 13 patients had high disease activity. Laboratory investigations of SLE group showed that antinuclear antibodies test was positive in 97.5% of the patients and the mean titer of anti-ds DNA was 154.8 ± 174.4 IU/ml [Table 2].
Table 2: Descriptive analysis of the studied cases according to laboratory investigations of patients with systemic lupus erythematosus (n=40)

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Lactobacilli and L. reuteri-specific bacterium DNA quantification was represented proportionally to the total bacterium DNA contained in the stool sample rather than as an absolute number. The relative abundance values of the different bacteria was displayed as follows: 4.75 × 10-5 was shown as 4.75E-05.

There was a statistically significant decrease in Lactobacilli relative abundance in patients with SLE (5.01E-03) compared with the control group (3.56 E-02), with P value less than 0.001 [Figure 1]. Regarding L. reuteri, there was also a statistically significant decrease in L. reuteri relative abundance in patients with SLE (5.51E-05) compared with the control group (1.78E-04) (P = 0.030) [Figure 2].
Figure 1: Box-plot analysis showing decreased Lactobacillus abundance among patients with SLE compared with the control group. SLE, systemic lupus erythematosus.

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Figure 2: Box-plot analysis showing decreased Lactobacillus reuteri abundance among patients with SLE compared with the control group. SLE, systemic lupus erythematosus.

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When studying the correlation between Lactobacilli and L. reuteri with different parameters (age, disease duration, C3, C4, and anti-ds DNA), no statistically significant correlation was detected, as shown in [Table 3]. However, an inverse but nonsignificant correlation between SLEDAI-2k scores for disease activity and Lactobacillus abundance (r=−0.198; P = 0.220) has been found.
Table 3: Correlation between Lactobacilli and Lactobacilli reuteri with different parameters in patients with systemic lupus erythematosus group (n=40)

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  Discussion Top


SLE is associated with loss of immunological tolerance as well as aberrant B-cell and T-cell functions. Moreover, a combination of genetic, epigenetic, and environmental factors induce it. New understanding of disease modulators, such as symbiotic bacteria, may allow fine-tuning of immune system components[16].

The gut microbiota's potential importance as a crucial aspect in the development of autoimmune disorders has lately attracted considerable attention. Changes in the composition of the gut microbiota are supposed to be participating in the etiopathogenesis of SLE, and several studies have been conducted on mice and humans to prove that gut microbiota dysbiosis affects the onset and progression of SLE[17].

In the current study, we found that Lactobacilli and L. reuteri were significantly lower in the guts of patients with SLE in comparison with the control group. These results come in agreement with a recent study by Gerges et al.[18] who found that the genus Lactobacillus was significantly lower in patients with SLE. This might explain the possible role of Lactobacilli and L. reuteri in the etiopathogenesis of SLE in several potential mechanisms including loss of tolerance and as consequence autoreactive T-cell or B-cell clones in addition to T-helper 17 cell growth and IFN-γ pathway stimulation.

Mu et al.[19] also discovered an obvious depletion of Lactobacillus in the intestinal microbiota of lupus prone mice in comparison with the control mice. Moreover, Zhang et al.[20] reported a significant decreased level of Lactobacilli in lupus-prone female mice with systemic autoimmunity, lymphadenopathy, and glomerulonephritis, which are like lupus-associated manifestations in humans compared with healthy control mice.

In the current study, there was an inverse but nonsignificant correlation between lactobacillus and SLEDAI-2k. This is in agreement with Gerges et al.[18]. Furthermore, Zhang et al.[20] stated that Lactobacillus was found to be negatively correlated with lupus activity, suggesting the usage of probiotics containing Lactobacillus to decrease the occurrence and/or severity of lupus flare.

Concerning the significant role of Lactobacilli and L. reuteri probiotics, many studies have been conducted to evaluate their beneficial role in modifying the disease activity. Tzang et al.[21] discovered that administration of mice with Lactobacillus paracasei and L. reuteri led to a significant increase in the antioxidant activity and reduction in cytokines such as IL-6 and TNF-α in severe lupus cases, suggesting that specific Lactobacillus strains can be used as a part of treatment strategy of patients with SLE.

Hsu et al.[22] revealed that supplementation of two L. reuteri strains, together with one L. paracasei strain, in lupus-prone mice suppresses lupus hepatitis through alleviating hepatic apoptosis and inflammation, and this may support the usage of L. reuteri as probiotics in liver disorders in cases of SLE. Moreover, Manirarora et al.[23] found that treatment of lupus-prone mice with three different strains of Lactobacilli (L. casei, L. reuteri, and L. plantarum) results in delaying lupus progression possibly via mechanisms involving T regulatory cell induction and IL-10 production, which may prolong disease remission.

Limitations of the current study include the following: first, relatively small sample size; second, only the lactobacilli genus was investigated, and this did not reflect the entire microbiome; third, being conducted at a single center and single ethnic group; and lastly, the dietary habits of study participants were not controlled, and it was better to be assessed using a food frequency questionnaire along with food diaries to minimize recall bias.


  Conclusion Top


Gut lactobacilli profile was altered among patients with SLE as our results showed significant decrease in abundance of lactobacilli and L. reuteri in SLE, and this might pave the way for potential therapeutic strategy through modulation of the gut microbiota. Furthermore, an inverse but not statistically significant correlation was discovered between Lactobacilli and SLEDAI-2k.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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