Study effect of probiotics and prebiotics on treatment of OVA-LPS-induced of allergic asthma inflammation and pneumonia by regulating the TLR4/NF-kB signaling pathway

Asthma, pneumonia, and lung injury are common acute respiratory problems in the clinic and allergic asthma is a relatively prevalent condition worldwide. Patients with asthma cannot be cured completely, and all available treatments generally aim to control the pathological condition. There is a huge economic burden on patients and governments [1, 2]. Allergic asthma is one of the most important health problems, especially in developed countries. Several pathways are involved in the pathophysiology of allergic asthma. Immune system dysregulation is directly related to health and is involved in the pathogenesis of a variety of immune disorders, including allergic diseases. On the other hand, the imbalance of inflammatory and anti-inflammatory factors and also dysregulation of the lung immune system through the TLR4/NF-kB signaling pathway can lead to pulmonary oxidative stress and injury [3, 4].

The therapeutic applications of probiotics and prebiotics in diseases such as asthma and pneumonia were not studied very well. Probiotics and prebiotics have been shown to possess immunomodulatory effects and be involved in the pathophysiology of various diseases. Short-chain (galacto-oligosaccharides) and long-chain (fructo-oligosaccharides or lcFOS) prebiotics along with probiotics (beneficial microbes) have also been noted to prevent allergic sensitization via regulating immune responses. Beneficial probiotic bacteria can modulate immune cells such as Th1, Th2, Th17, Treg, and also B cells [5, 6].

There are many microbial infections can active TLR4 signaling and probiotics as part of commensal gut microbiota, can have effect on the TLRs, especially TLR4 [7, 8]. LPS is structural material of bacteria and TLR4 activation by bacteria is initiated via LPS/TLR4/NF-kB pathway [9, 10]. On the other hand, LPS can induce acute lung injury and inflammation, which leads to accumulation of inflammatory cells in the lung and airway [11, 12]. OVA can induce allergic asthma model but, OVA-LPS can induce asthma and acute inflammation in airways. Also, it can involve with TLR4 and actives TLR4 signaling. Therefore, in this study, we explored the functional mechanisms of probiotics and prebiotics in regulating of the host’s defense against LPS-OVA-induced lung injury and airway inflammation and the TLR4/NF-kB pathway. Also, immuno-inflammatory responses and pulmonary pathological features were studied in the mice that were treated with probiotics and prebiotics. The effect of probiotics and prebiotics of on acute form of allegro-inflammatory airway asthma with TLRs involving was studied.

Atopy and allergy affect more than 30% of the global population. The prevalence of allergic diseases has been increasing in developed countries during the past 20 years and probiotics, as live microorganisms, have been beneficial for managing allergic diseases [5]. Clinical evidence showed the therapeutic role of probiotics in allergic rhinitis and asthma, improving cure rates and alleviating allergic rhinitis symptoms and the need for drug use. In clinical trials, receiving probiotics has led to the successful control of the immunologic outcomes of allergy (total and allergen specific IgE, leukocyte counts, blood and nasal eosinophils, Th2/Th1 ratio, IL-4 level, thymus-and activation-regulated chemokines, and quality of life). Also, probiotics increased the number of Treg and TGF-β-producing cells; on the other hand, Treg activity could modulate the severity of allergic inflammation [5, 6]. In general, probiotics can decrease disease severity mainly via modulating the immune responses involved in the development of allergic inflammation [5, 15]. According to hygiene hypothesis, in early childhood, lack of exposure to pathogens increases the susceptibility to allergic diseases such as asthma. It leads to Th1-like pattern immunity shits to the Th2-immunoallergic response. Probiotics in adequate amounts confer a health benefit to the host and have ability to modulate mucosal immune responses, stimulatory effects on the innate and adaptive immune system and enhance mucosal barrier functions [16, 17]. Moreover, the innate immune stimulation by bacteria is essential for the establishment of mucosal immune tolerance and, it can be continued by TLR2, TLR4, and TLR9 recognizing microbial components via production of Th1 cytokines. Probiotics may have a strong action in allergic pathologies via TLRs pathways. In particular, the probiotics play a pivotal role in the Th1/Th2 balance. In the allergic asthma, probiotics attenuate the influx of eosinophils to the airway and reduce the levels of MCP-1, and IL-13 level [16, 17]. We observed that probiotic and prebiotic treatments attenuated the recruitment of activated eosinophil to the lung and decreased EPO activity in BALF. Probiotic treatment also decreased eosinophils’ infiltration into BALF. The increased neutrophils in BALF of the LPS challenged groups was controlled by probiotics and prebiotics. LPS acts as TLR4 activator that in this study, increased and recruited neutrophils to the BALF and treatment with probiotics and prebiotics could markedly decrease neutrophils infiltration to the BALF. Therefore, TLR4 was involved in this study by LPS and effect of probiotic and prebiotic treatments on inflammation via TLR4 was noted. Probiotic and prebiotic treatments diminished the production of TSLP, cyst-LTs, LTC4, LTB4, GTP, and GOT which lead to control of inflammation in airways and harnessing of immune-inflammatory responses in lung and could control bronchial inflammo-pathology. Also, Probiotic and prebiotic treatments decreased the elevated levels of total IgE, OVA-specific IgE, and IgG₁ and had effect on humoral immunity. The B cell activation is mainly controlled by T cells and T cell releasing cytokines. When B cells are activated can produce and release specific immunoglobulins. Allergen-activated B cells produce IgG₁, total and OVA-specific IgE (in mice) and produced Igs bind to the specific receptors on surface of the mast cells, lead to mast cells degranulation and releasing of allegro-inflammatory mediators [2, 4]. In this study, treatment by probiotic had effect on T cell's cytokines and T cell response (cellular immunity) and may have directly effect on B cell to decrease of allegro-inflammatory immunoglobulins. Between two used treatment (probiotic and prebiotic), probiotic had stronger effect on control of allegro-inflammatory factors than prebiotic and could significantly decrease asthma related immune-inflammatory biomarkers. These results implicated that probiotics might have anti-inflammatory properties. In asthmatic lung tissues, the infiltration of inflammatory cells into perivascular and peribronchial areas and increased scores of goblet cell hyperplasia and mucus secretion were observed. This inflammation significantly decreased in probiotic- and prebiotic-treated animals. The scores of goblet cell hyperplasia and mucus secretion reduced in probiotic-treated mice.

On the other hand, according to the ‘hygiene hypothesis’, the increasing prevalence of immune-mediated anti-microbial activity may be related to the decreased incidence of allergic diseases. The crosstalk between gut microflora (probiotics) and the immune system (gut-associated lymphoid tissues) enables balancing Th1/Th2 immune responses in healthy individuals and mediates tolerance and mucosal immunity [6, 15, 17]. The effects of probiotics and prebiotics on Th2-related pro-inflammatory cytokines (IL-4, IL-5, IL-13, IL-25, and IL-33), Th1-related pro-inflammatory cytokine (IFN-γ) and Th17-related cytokine (IL-17) were analyzed. Probiotic and prebiotic treatments reduced the levels of Th2 cytokines (IL-4, IL-5, IL-13, IL-17, IL-25, and IL-33) and increased IFN-γ level in the BALF of asthmatic mice as compared to the non-treated asthma group. Also, gene expression of IL-8 was decreased, and IL-38 was increased in the probiotic- and prebiotic-treated groups compared with the non-treated asthma group, indicating the regulatory effects of probiotics and prebiotics on the Th1/Th2 balance. In this study, probiotic had surprising effect on modulation of cytokines levels in treatment of asthma, but prebiotic treatment had weak effect especially on cytokines levels and genes expression. It was reported that bacterial probiotics (Bifidobacterium infantis) treatment decreased levels of OVA specific-IgE, and IgG in serum, reduced the release of IL-4, -5, -13 in splenocytes. Moreover, the protective effect of probiotics on allergic asthma was correlated with superoxide dismutase and antioxidative enzyme [19, 20]. Treg cells have important role in asthma, and less functional Treg cells lead to enhanced Th2-type immune responses, and eosinophilic airways inflammation. High-fibre prebiotics, via the acetate production, prime Treg cell to protect against asthma. Prebiotics such as soluble fibres and inulin should be fermented by beneficial bacteria and this is time-taking process. Therefore, these products cannot quickly act and present anti-inflammatory properties [13, 21].

Several studies showed that prebiotic supplementation improved airway hyperresponsiveness and decreased inflammatory cells’ counts in the sputum of asthmatic patients [13, 22‐24]. Penh is a dimensionless index that generally evaluates changes in the shape of the airflow pattern entering and leaving a whole-body flow plethysmograph when an animal breathes and also by monitoring of AHR changes via connected tube to the ventilator. A higher level of Penh indicates a more severe bronchospasm/airway hyperreactivity to allergens. Interestingly, the oral administration of probiotics and prebiotics could improve AHR. Our results revealed that probiotic administration, but not prebiotic, significantly decreased AHR in asthmatic animals. Nevertheless, prebiotic treatment also reduced AHR, but the results were not significant that was similar to a study in 2020 [25].

LPS as part of the cell membrane of Gram-negative bacteria is one of TLR4 ligands. When LPS binds to TLR4, activates the MyD88 leading to the downstream production of kinases, which allow translocation of the NFκB-related proteins into the nucleus. The NF-κB complex triggers transcription of genes that are involved in the immune-inflammatory responses, and once activated begins a cascade of pro-inflammatory cytokine, specifically TNF-α, IL-1β and IL-6. Once activated NLRP3 inflammasome via microbial components cleaves pro-IL-1β, -IL-18, leukotrienes and prostaglandins [26‐28]. In this study, LPS was used as TLR4 activating factor for airway inflammation. When TLR4 in involved, it can participate in asthma pathophysiology and treatment with probiotic has influence on the TLR4 and related signaling pathways. Therefore, the effect of probiotic on regulation of TLR4/ NF-kB Signalling Pathway will be notable. In this study, it was observed that probiotic therapy could decrease and control gene expression of TLR4, NLRP3 and NF-kB as main inflammatory molecules.

It has been demonstrated that many factors and signaling molecules contribute to the induction of allergic inflammation, including cytokines, chemokines, superoxide, ECP, TLRs, and intracellular kinases (NF-κB, ERK, Akt, p38, MAPK, etc.) [3, 29, 30]. AngII could increase ROS production and oxidative stress by activating NADPH oxidases, and promoting release of inflammatory cytokines. It was demonstrated that AngII exhibits pro-inflammatory responses via TLR4 upregulation and stimulating TLR4-dependent signaling pathways. Likewise, AngII upregulates TLR4 and MyD88 gene and AngII-TLR4 crosstalk promotes pro-inflammatory effects, and also, AngII-induced ROS production is dependent on the presence of a functional TLR4. AngII-induced TLR4 upregulation promotes functional consequences such as increase in NF-kB activation, which is dependent the presence of functional AP-1, ETS, and PU.1 transcriptional site. Moreover, molecular mechanisms of AngII-TLR4 activation may indicate an interaction between TLR4 and AT1r signaling downstream effector molecules. Also, TLR4-mediated NF-kB activation in response to LPS is dependent on EGFR signaling. These suggest a regulatory mechanism between AT1r, TLR4, and NF-kB indicating their role in pro-inflammatory effects [31, 32]. Moreover, AngII can directly activate the NF-κB that is critical for initiating the inflammatory cytokines responses during the inflammation. Also, during the inflammatory process, activation of the TLR4 could induce the pro-inflammatory cytokines production and activation of NF-κB via MAPK signaling pathway. On the other hand, TLR4 expression is modulated by local AngII and caused the activation of NF-κB signaling and induction of TNF-α, CD40, and IL-6 expression (33). In this regard, probiotics were used in this study to modulate these pathways. Probiotic and prebiotic treatments reduced the expression of asthma related genes involved in the PI3K/Akt and TLR4/NF‐κB signaling pathways in asthmatic lungs. As we observed, the expression levels of Akt, PI3K, NF-kB, and TLR4 raised in asthmatic mice, and the administration of probiotics dramatically attenuated the expression levels of these genes. The oral administration of probiotics and prebiotics remarkably inhibited the gene expressions of TLR4, NLR3, NF‐κB, and MyD88, but decreasing of TLR4 gene expression by prebiotics was not significant.

IL-4 induces GATA3 expression and in result Th2 cell differentiation. OX40 ligand is linked to the development of Th2-related allergic responses. The activity of probiotic (Lactobacillus murinus) against food allergy showed that oral administration attenuated allergen-induced diarrhoea, mast cell activation, serum IgE and IL-4 production by splenocytes. It enhanced IFN-γ and IL-12 production. Concordantly, it suppressed expression GATA3 and OX40 ligand expression by intestinal CD11c + cells and increased T-bet expression. These demonstrate that probiotic possesses anti-allergic activities via modulating CD11c + cell functionality and the Th1/Th2 immunobalance, which may be employed against food allergy [34, 35]. IL-38 acts as a partial antagonist receptor of IL-36 inflammatory cytokine, suppressing the asthma-induced production of CXCL8 and inhibiting allergic inflammatory responses [36‐38]. IL-38 may regulate airway inflammation and can diminish the production of the inflammatory cytokines and chemokines that are regulated by the intracellular p38, ERK1/2, and NF-κB signaling pathways [39, 40]. Eotaxin, a chemotactic cytokine/chemokine with potent effects on eosinophils, increases in asthmatic lungs after exposure to allergens, causing the accumulation of eosinophils in airways. After treatment with both probiotics and prebiotics, the gene expression of eotaxin, CCL11, and CCL24 reduced in the lungs of asthmatic mice, but only CCL11 gene expression by probiotic was significant.

Bronchial inflammation is principal problem in asthma and free oxygen radicals have main role in airway inflammation and pathogenesis of allergic asthma. The oxidation and antioxidative defense modulation is new therapy in management of asthma. Mitochondrial abnormalities have collaboration with oxidative stress that can cause airways injury. Co-Q10 is one of the important antioxidant supplementations, which contributes in the regulation of mitochondrial function and pathology, and also, allergic inflammation. CO-Q10 can induce resistance against the asthma development, protect upper and lower airways against allergic response, and control inflammation [32, 41]. The effects of probiotics on the immune system are classified into two categories involving the activation of the innate immune cells and the inhibition of excessive abnormal immune responses [42, 43]. Oxidative stress is involved in the pathogenesis of several tissue damages and expression of striatal proteins contribute to oxidative stress and damage. ROS production and subsequent toxic events including; mitochondrial membrane potential collapse, lipid peroxidation, lysosomal membrane injury and enzymes dysfunction indicating that oxidative stress. Mitochondrial ROS formation is important mediator in tissue damage and antioxidants combat with free radicals to control oxidative stress. Also, angiotensin II is produced by ACE and can bind cell-surface receptors via AT1 and type 2. AT1 receptor is linked to ROS formation, and inflammation via activation of several downstream signals including, Ras/Rho, ERK, MAPK and JAK/STAT signaling pathway [44, 45]. Probiotics and prebiotics can shift immune reactions from Th2 to Th1 responses in allergic diseases, promoting tolerance in allegro-inflammatory conditions. Probiotics also modulate cellular (Th1, Th2,) and humoral (B cells) immune responses and improve general health by preventing allergies and immune disorders. Probiotic could reduce oxidative and inflammation markers and control airway inflammation and asthma pathophysiology, but prebiotic could not strongly control the inflammation and allergic asthma symptoms. Probiotics use major mechanisms to improve allergic asthma clinical symptoms and prevent them, with several known mechanisms: Th2 suppression and shift response to Th1, tolerance induction, increase of IL-10, Treg cells and their responses for control immune-inflammation, increase the IFN-γ/IL-4 ratio, decrease eosinophil, Th2 cytokines and IgE levels, reduce gene expression of allegro-immune response related, chemokines and cytokines signaling pathways, and also, control AHR. Finally, probiotics can be used as therapeutic approach to treatment of allergic asthma with priority of the tolerance and the lowest side effects.

The probiotics act through several mechanisms to maintain the homeostasis and perform beneficial functions, including pathogens elimination, vitamins and short-chain fatty acids production, as well as modulation of the systemic and mucosal immune system and also inflammation via controlling of NF-κB activation and pro-inflammatory cytokines. Probiotics are live organisms can act as delivery systems and proliferate in tissues for long time, but prebiotics have not these characters. Also, mechanisms action of probiotics in the host include; colonization, regulation of a dysbiotic microbiota, epithelial barrier protection by maintaining tight junction integrity, competitive exclusion of pathogens, production of anti-inflammatory properties, signaling molecules regulation of immunological pathways and inducing host autophagy to attenuate oxidative stress-induced injury. Prebiotics may be added as supplementary agent and stimulate probiotic action. Prebiotic fermentation can modulate the composition and function of probiotics. Prebiotics with regulatory ability, modify the commensal microbiota to a beneficial state. Prebiotics, when associated with a probiotic, are more efficient than when are used separately (13, 14, 46). Therefore, prebiotics may show lower effect than probiotics on control of eosinophilic airway inflammation, EPO activity, immune-allergic response, and allergic asthma.

In this study, there were some limitations. We did not study the effect of probiotic and prebiotic on control group and also on the OVA group and LPS group. We did not have data about protein of the signaling pathways, and phosphorylation of pathway proteins. We could not study to verify the pathway using genetic gain/loss of function models or pharmacologic treatments. We could not show that probiotics can inhibit asthma pathophysiology in absence of TLR4. Also, we did not study the effect of probiotic and prebiotic on chronic form of airway inflammation and asthma.