Targeting the gastrointestinal system as a site for regulation of immune dysfunction has long been considered key to moderating an aberrant immune response. This is especially pertinent in autoimmune conditions and for the relief of atopic conditions, including eczema and rhinitis. Two clinically trialled probiotic strains, Lactobacillus paracasei (LP-33^®) and Lactobacillus rhamnosus GG (LGG^®), help restore immune control and moderate over-active immune responses. These strains can be used during all stages of life including prenatally, in infants, children and adults, and is intended for safe long-term use.

Figure 1: One of the mechanisms by which probiotics enhance immune control is through the production of regulatory T-cells.^[1]

Background Technical Information

Gut-Immune Crosstalk

Microbial diversity in the gastrointestinal tract (gut) is important for many aspects of human health, with perturbations of intestinal host-microbe interactions leading to defects and altered host responses, metabolically, structurally and protectively.^[2] This increases the risk of pathogenic processes that can promote a range of disorders, including atopy^[3] and autoimmunity.^[4]

Probiotics, defined as ‘live microbial ingredients that, when ingested, confer health benefits on the host’,^[5] have been shown to influence the development, maturation and maintenance of the crosstalk between the gut microbiota.  The gut microbiota will, in turn, crosstalk with the innate and acquired immune systems via the interaction of their components and/or metabolites.^[6] 

Such interactions include, but are not limited to, a direct impact on the intestinal epithelium, including the gut associated lymphoid tissue (GALT) and intestinal lamina propria, T cell polarisation for the production of the appropriate immune reaction, and the regulation of endogenous microbiota.^[7],[8],[9] Probiotics also enhance the gut microbiota by replenishing levels of beneficial bacteria and inhibiting the growth of more pathogenic flora. This is achieved via the occupation of receptor sites and competition for nutrients, referred to as competitive exclusion.^[10],[11] These mechanisms form the basis for the oral supplementation of probiotics, which ultimately aims to increase the relative numbers of beneficial bacteria colonising the gastrointestinal tract. This increase contributes to an individual’s overall wellbeing.

Allergy Prevalence

Allergies are the fastest growing group of chronic diseases, with Australia and New Zealand having the highest prevalence in the developed world. Those most affected are the working age population, with 78% of people with allergies aged 15 to 64 years.  Overall, allergies affect almost 20% of the Australian population.^[12] If current trends continue, it is predicted that by 2050 the number of Australians affected by allergies would increase by 70% to 7.7 million people.

Autoimmune Prevalence

Autoimmune disease presents a significant health issue in Australia and New Zealand, with approximately one in twenty people affected.^[13] Some of the common autoimmune conditions, which represent a significant chronic health burden in Australia, include inflammatory arthritides such as rheumatoid arthritis (RA), along with Hashimoto’s thyroiditis, coeliac disease, inflammatory bowel disease (IBD), multiple sclerosis (MS), systemic lupus erythematosus (SLE) and type 1 diabetes.^[14]

Actions

T Regulatory, Th1 and Th2 Modulation

GALT is a central player in host defence. A highly organised system of immune structures, the GALT includes mesenteric lymph nodes, Peyer’s patches, isolated lymphoid follicles and fat-associated lymphoid tissues, all of which facilitate the interactions between antigen presenting cells (APCs) and lymphocytes and their subsequent activation and differentiation.^[15]

The extensive antigenic sampling within the GALT enables the interaction between antigen-specific B and T cells, resulting in an adaptive immune response where T cells play a critical role. The main types of T cells currently recognised are T helper (Th) cells including type-1 or Th1, Th2, Th9, Th17, Th22, T regulatory cells (Tregs) and follicular helper T cells (Tfh).^[16] Transition between the types of T cells occurs in the gut and is an important means of maintaining gut homeostasis.^[17] 

Intestinal macrophages induce the action of the Tregs which, as the name suggests, play an important role in the regulation of the immune system. Tregs are essential for dampening inflammation in autoimmune and allergic disorders alike. Tregs down-regulate excessive levels of all aforementioned T helper cells and are critical in the maintenance of immune tolerance. Tregs express the key immunoregulatory cytokines IL-10 and transforming growth factor-beta (TGF-β), both of which significantly decrease inflammation.^[18]  The synergistic combination of LGG^® and LP-33^® have been shown to induce Treg production,^[19],[20] providing important immunoregulatory support.

LGG^® increases the secretion of IL-10, leading to an up- regulation of the development of B cells and down-regulation of immunoglobulin-E (IgE) synthesis.^[21] IgE-driven inflammation is apparent in conditions such as asthma and rhinitis.^[22] This regulation of the B cells and IgE demonstrates an immunomodulation response sufficient to counter the excessive inflammation associated with up-regulated Th2 activation, once again seen in allergy driven conditions such as asthma, allergic rhinitis and atopic dermatitis.^[23] This positive effect has been shown in children and adults alike.^[24],[25]

Allergic rhinitis is a condition characterised by excessive Th2 activity and the consequent inflammatory response. The gut microbiota interaction with GALT moderates inflammation and immune reactivity in allergic rhinitis. LP-33^® and LGG^® have been shown to down-regulate the production of IgE and suppress the Th2 response via the inhibition of IL-4 secretion.^[26],[27] 

Low levels of the Th1 cytokine, interferon gamma (IFN-γ), is associated with cow’s milk allergy. In a randomised, double-blind study design, 5 billion colony forming units (CFU) of LGG^® were given daily, along with concomitant use of an elimination diet and skin treatment. This led to an increase in IFN-γ production of peripheral blood mononuclear cells in infants with cow’s milk allergy and in infants with IgE-associated dermatitis. This beneficial study not only showed the Th1 immunomodulatory signalling potential but also the importance of probiotic strain specificity, as LGG^® in combination with three other bacteria (another strain of L. rhamnosus, Bifidobacterium breve Bbi99, and Propionibacterium freudenreichii ssp shermanii JS) did not yield these positive effects.^[28]

Modulates Inflammation and Autoimmune Reactions

The relationship between gut dysbiosis, a condition where the natural microbial balance is disrupted in a certain part of the body,^[29] and inflammation is well established. Environmental factors such as environmental contaminants, increased intake of advanced glycation end products and food additives, circadian disruptions and stress all contribute to dysbiosis.^[30] Concurrent defects in the host’s immune response may result in the accumulation of intestinal commensal bacteria that have the capacity to locally invade the intestinal mucosa and trigger an abnormal inflammatory response.^[31] Dysbiosis results in a loss of protective bacteria and/or increased accumulation of pathogenic agents, leading to chronic inflammation involving hyperactivation of Th1 and Th17 cells and concurrent reduction of Tregs.^[32] The image on the right hand side in Figure 2 highlights this using the example of the highly inflammatory condition, IBD.

Autoimmune disease is influenced by the diversity of the gut microbiota, as highlighted in Figure 3. The presence of segmented filamentous bacteria (SFB)[*], for example, induces Th17 development in the intestine. Increased levels of Th17 enhance the expansion of pathogenic autoantigen-specific T cells in the intestine and cause inflammation in the central nervous system (CNS) leading to multiple sclerosis, for example. Another example is microbiota-induced interleukin 1β (IL-1β) signalling, which participates in the development of RA through the induction of Th17 cells.^[33]

Figure 2: Protective and pathogenic role of gut microbiota in a case of autoimmunity.^[34]

Figure 3: Influence of gut microbiota on the pathogenesis of autoimmune and allergic disease.^[35]

In allergy-driven inflammation there is a bias toward an excessive Th2 response, with allergic individuals more likely to show gut dysbiosis than those who are not.^[36] Restoration of the gut microbiota results in an increase in Th1 and Th17 cells and a reduction of Th2 levels, indicating the important role of the gut microbiota in maintaining the balance of T helper cell responses.^[37]

Clinical Applications

Reduce the Prevalence and Intensity of Atopy

As noted above, atopy (allergy) is associated with an imbalance in T cell production; with Th2 cell production predominating and leading to an increased production of histamine-producing IgE.^[38] Resulting conditions include hayfever, allergic rhinitis, atopic dermatitis/eczema and dust mite allergy. As previously mentioned, LGG^® has been shown to increase the secretion of IL-10, in turn up-regulating the growth of B cells and down-regulating IgE synthesis.^[39] This immunomodulatory response is sufficient to counter the excessive inflammation associated with up-regulated Th2 activation and the resulting atopy.

A randomised controlled trial was conducted to assess the efficacy of LP-33^® in patients with persistent allergic rhinitis to grass pollen, who were currently taking an antihistamine and presented with altered quality of life. Four hundred and twenty-five patients received either LP-33^® (2 billion CFU/day) or a placebo, plus an antihistamine for five weeks. The primary outcome was the improvement in Rhinitis Quality of Life (RQLQ) global score and the secondary outcomes included nasal and ocular symptoms. The RQLQ global score decreased significantly in the LP-33^® group (Figure 4) with significant differences in ocular symptoms also being reported.^[40] This study highlights the effectiveness of a strain-specific probiotic as an adjuvant to medical treatment for atopy, demonstrating a significant improvement in patients’ quality of life.

Figure 4: Reduction in RQLQ global score with LP-33^® compared to placebo.^[41]

LP-33^® has also been shown to improve the quality of life for those living with perennial allergic rhinitis triggered by house-dust mite allergy. A total of 90 subjects were enrolled in a placebo-controlled trial and assigned to two groups. The first group received LP-33^® (10 billion CFU/day) for 30 days, while the second received a placebo. Subjects completed a rhinoconjunctivitis symptom questionnaire during each clinical visit. The overall symptom score was significantly reduced for the LP-33^® group, while the placebo group saw an increase in scores, including the frequency and ‘level of bother’ after the 30-day treatment (Figure 5).^[42]

Figure 5: Changes in symptom scores of subjects with allergic rhinitis induced by house-dust mite after taking LP-33^® for 30 days.^[43]

A randomised-controlled trial was conducted in 159 mothers who had at least one first-degree relative or partner with atopic eczema, allergic rhinitis, or asthma. LGG^® (20 billion CFU/day) or placebo was given to expectant mothers from 24 to 28 days (mean 26 days) prior to expected delivery and for six months postnatally, either to the breastfeeding mothers or directly to the infants. The frequency of atopic eczema in the probiotic group was 50 percent that of the placebo group, 23% versus 46% (Figure 6), with the preventive effect impacted by the mode of administration.^[44]

A subgroup of this study included mothers who breastfed and took the probiotic themselves prenatally and for at least three months postnatally.  The infant cord blood IgE levels and the levels of breast milk TGF-β2 was assessed at birth and three months postnatally, respectively. TGF-β2 is an anti-inflammatory factor and provides a measure of the immunoprotective potential of the breast milk. In these 62 mother-infant pairs, administration of probiotics to the pregnant and lactating mother increased TGF-β2 levels in the mothers, compared with those receiving placebo (2885 pg/mL vs. 1340 pg/mL). The risk of developing atopic eczema during the first two years of life was significantly reduced in infants of the mothers who received probiotics, in comparison with the risk in infants whose mothers received placebo (15% vs. 47%) (Figure 6).

The preventive effect of LGG^® on atopic eczema has also been shown to extend beyond infancy. The aforementioned study was followed up at four years and again at seven years. At four years (107 children assessed), there was a reduced incidence of atopic eczema in the probiotic group.^[45] These effects continued through to the final follow-up visit at seven years of age. One hundred and sixteen children completed the seven-year follow-up: 62 of 82 (76%) in the placebo group and 53 of 77 (69%) in the LGG^® group. Overall, the risk of eczema during the first seven years of life was significantly reduced in the LGG^® group compared with the placebo group (Figure 7).^[46]

Figure 6: Treatment effect of LGG^® vs. placebo on atopic disease at 2 years. (Bars are 95% CI).^[47]

It should be acknowledged that a study investigating the effects of LGG^® on the prevention of atopic dermatitis, taken by pregnant women and then by breastfeeding mothers or formula-fed infants, failed to find a beneficial effect for LGG^®. However, the dosage of LGG^® used in this study was 5 billion organisms twice daily, half that of the primary study above, suggesting that the lack of positive results could be due to an insufficient dose.^[48]

Figure 7: Kaplan-Meier curves for the proportion of children without eczema at the ages of 2, 4, and 7 years in LGG^® (n = 64) and placebo (n = 68) groups (p=0.008), indicating a protective effect of LGG^® on the risk of developing atopic eczema.^[49]

Autoimmune Conditions

A randomised-controlled study was conducted with 21 patients with mild RA to assess the potential impact of LGG^® on arthritic symptoms. Patients were randomised to receive LGG^® (10 billion CFU) or a placebo for 12 months. Arthritis activity was evaluated by clinical examination, a Health Assessment Questionnaire (HAQ) index, and laboratory tests for the inflammatory markers erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), pro- and anti-inflammatory cytokines.^[50] The results showed the mean number of tender and swollen joints decreased from 8.3 to 4.6 in the LGG^® group and only 5.5 to 4.8 in the control group (Figure 8).^[51]

Figure 8: Reduction in number of tender and swollen joints in RA patients on LGG^® compared to placebo.^[52]

In a small pilot study, four children with mild to moderate Crohn’s disease were given LGG^® (20 billion CFU/day) for six months. Outcomes measured were changes in intestinal permeability and the paediatric Crohn’s disease activity index. The results showed that at four weeks mean paediatric Crohn’s disease activity index scores were 73% lower than baseline with a corresponding improvement in intestinal permeability.^[53]

A study was conducted to assess the efficacy of LGG^® as maintenance treatment in patients with inactive ulcerative colitis (UC). One hundred and eighty seven patients were randomised to receive either LGG^® alone (18 billion CFU/day), LGG^® in combination with mesalazine or mesalazine alone. Disease activity was measured at baseline, 6 and 12 months and the primary outcome was sustained remission. The percentage of patients maintaining clinical remission after 6 and 12 months of treatment was 91% and 85% respectively for the LGG^® group, 87% and 80% for the mesalazine group and 94% and 84% for the combined treatment group.^[54] The study demonstrated that LGG^® significantly prolonged the relapse-free time in patients with UC compared with mesalazine alone, thus potentially improving patients’ quality of life.^[55]