Probiotics and Amelioration of Rheumatoid Arthritis: Significant Roles of Lactobacillus casei and Lactobacillus acidophilus

Rheumatoid arthritis is a chronic autoimmune disorder that can lead to disability conditions with swollen joints, pain, stiffness, cartilage degradation, and osteoporosis. Genetic, epigenetic, sex-specific factors, smoking, air pollution, food, oral hygiene, periodontitis, Prevotella, and imbalance in the gastrointestinal microbiota are possible sources of the initiation or progression of rheumatoid arthritis, although the detailed mechanisms still need to be elucidated. Probiotics containing Lactobacillus spp. are commonly used as alleviating agents or food supplements to manage diarrhea, dysentery, develop immunity, and maintain general health. The mechanism of action of Lactobacillus spp. against rheumatoid arthritis is still not clearly known to date. In this narrative review, we recapitulate the findings of recent studies to understand the overall pathogenesis of rheumatoid arthritis and the roles of probiotics, particularly L. casei or L. acidophilus, in the management of rheumatoid arthritis in clinical and preclinical studies.


Introduction
Rheumatoid arthritis (RA) is an autoimmune disorder characterized by swollen joints with chronic pain, stiffness, reduced functionality, osteoporosis, and cartilage degradation leading to a situation of disability [1,2]. RA is prevalent in 0.3-1.0% of adults aged 20-40 years globally, and it is very common among older adults aged 75 years or above [3,4]. Women are relatively more susceptible to RA [4]. RA patients experience age-related comorbidities such as diabetes, cardiovascular disorders, nephritis, chronic obstructive pulmonary disease (COPD), psychological distress, osteoporosis, asthma, and cancer, leading to a higher mortality rate [5].
RA is currently treated with nonsteroidal anti-inflammatory drugs (NSAIDs), as these drugs prevent prostaglandin synthesis by inhibiting the cyclooxygenase enzymes (COX-1 and COX-2) and thereby act against inflammation and pain. Drugs in this group include paracetamol, ibuprofen, naproxen, diclofenac, indomethacin, ketoprofen, meloxicam, and many more [6]. Besides NSAIDs, disease-modifying anti-rheumatic drugs like methotrexate

RA and Gastrointestinal Tract
The gastrointestinal tract is connected with the environment, and it is an essential getaway for the intake of beneficial and harmful microbes and their metabolites through food and drinks. The human gut harbors over 100 trillion bacteria, and these microbes Several genetic, environmental, and socioeconomic factors are associated with RA initiation and progression [44]. Fat-rich and low-vegetable diet, smoking, gut microbiota, periodontal diseases, bacterial or viral infections lead to different inflammatory conditions and results in RA [47][48][49] (Figure 1). RA is also influenced by gender as women are more susceptible to RA than men and several factors influence the disease, such as hormonal deficiency (estrogen deficiency in pre-and post-menopausal women), genetic factor (Xchromosome related), the prevalence of depression, neuropathic pain, fibromyalgia and osteoporosis (in post-menopausal women) [50][51][52][53]. Apart from environmental factors, increased citrullination is also observed in alveolar macrophages in smokers' lungs, and thus smoking may involve the pathogenesis of RA [47].
Citrullination is a peptidylarginine deiminase-mediated enzymatic conversion of arginine residues of protein to citrulline residues, and dysregulation of the peptidylarginine deiminases enzymes causes increased citrullination of protein in the joints of RA patients [54].

RA and Gastrointestinal Tract
The gastrointestinal tract is connected with the environment, and it is an essential getaway for the intake of beneficial and harmful microbes and their metabolites through food and drinks. The human gut harbors over 100 trillion bacteria, and these microbes have a potential role in the digestion, metabolism, nutrition, disease control, and maintenance of general well-being [55][56][57][58]. The epithelial cell barrier and intestinal single cell layer determine the rate of entry of microbes or antigens into the bloodstream [59,60]. Disturbance in the gastrointestinal homeostasis, especially massive changes in gut microbiota composition, results in diarrhea, dysentery, or inflammatory responses such as RA [61][62][63][64]. Increased bacterial lipopolysaccharides in the bloodstream and their deposition in the synovial fluids can cause proinflammatory responses (by releasing cytokines) and RA [65,66] (Figure 1).
Besides genetic, environmental, and physiological changes like citrullination of proteins and deposition in the joint tissues, RA is also believed to be caused by gut bacteria like Prevotella. Prevotella spp. are anaerobic, non-spore-forming bacteria, and these are part of normal gut flora. Prevotella contributes to polysaccharides breakdown, and their presence is relevant to the consumption of carbohydrate and high fiber diets [48]. P. copri is also responsible for the generation and progression of intestinal dysbiosis in people at their early stages of RA or risk on RA development [67][68][69]. Increased Prevotella spp. was found in the intestines of people carrying genotype of RA before developing clinical symptoms of the disease [70]. Increased abundance of P. copri and its antibodies are detected in patients with RA [71]. The increased population of P. copri in the intestinal lumen probably enables it to defend itself against myeloid cells and T cells that leads to immune responses [69] ( Figure 1). Prevotella also activates the TLR-2 receptor of the intestinal epithelial cells and stimulates the release of proinflammatory cytokines such as IL-1β, IL-6, and IL-23 and promotes the activation of Th17 cells that leads to massive production of IL-17, inflammation, and initiation of RA [72] (Figure 1).
A meta-analysis study showed that probiotic supplement helps reduce IL-6 levels in RA patients, which may help to manage RA. However, it did not reduce DAS scores of RA patients, and it may not be effective in established RA patients [73]. It is known that gut dysbiosis favors increased population growth of Eggerthella lenta or Collinsella aerofaciens bacteria in the gut [74]. A clinical study showed decreased population of Faecalibacterium (a Firmicutes bacteria abundant in healthy human gut) in people experiencing RA [75] (Table 1). Faecalibacterium is responsible for butyrate production [76] that stimulates mucin secretion and lubrication of inner gut epithelium. As the Faecalibacterium population decreases, the gut is more vulnerable to opportunistic organisms like Collinsella and Eggerthella. Collinsella induces the release of cytokines (e.g., IL-17α) and chemokines (e.g., CXCL1 and CXCL5) that contribute to RA development by activating NFkB and neutrophils [75]. Eggerthella causes the citrullination of proteins that contributes to RA development. RA-associated decreased population of Streptococcus and Haemophilus are also observed in the human gut with the escalation of Prevotella histicola and P. oulorum numbers [77]. Higher fecal Lactobacillus casei was also reported in RA patients [78]. Thus dysbiosis-related changes in Collinsella aerofaciens, Eggerthella lenta, Faecalibacterium spp., Haemophilus spp., Prevotella spp., and Streptococcus spp. population induce loss of integrity of inner epithelium of gut and development of RA [75,77] (Table 1).  [91][92][93] The situation regarding the enteric biome is complicated by the presence of pathogenic and commensal enteric viruses (forming the enteric virome) and their effects on intestinal homeostasis and immune responses. Since the subject matter is still challenging like experimenting with mice infected with commensal enteric human viruses is still not a feasible experiment that has been done [94], and since existing literature lacks reports (to our knowledge) on any enteric viral biome effect on initiation and development of RA, this review will confine itself to enteric microbial biome (meaning enteric bacteria and does not take into account virome or fungal biome) and any disturbances in their homeostasis leading to RA. The main objective of this review is to take a close look at whether the administration of probiotics in the form of Lactobacillus casei and Lactobacillus acidophilus can cause amelioration of the RA. However, it is possible that disturbances in the pattern recognition receptors (PRRs) and their signaling pathways can lead to disruption of immune homeostasis, and which might act as a trigger for RA.
It has been reported that the virome can have a profound influence on the composition and functional properties of the microbial biome, and in the practical sense, it alters the development and function of the immune system [95]. It remains for future studies to determine whether the predominance of Prevotella spp. in the human gut leading to RA is a result of gut virome alterations and whether taking probiotics like Lactobacillus can help the intestine to revert back to normal virome homeostasis. The question of gut inflammation, immunity, and the role of gut virome has been raised before [96], but any answer is yet to be found, and the topic falls outside the scope of this review. A very recent pre-print article reports dysbiotic oral and gut viromes in treated and un-treated RA patients [97]. Despite recent advances on gut virome and resultant pathogenesis if the homeostasis is disturbed, there is virtually no information on the role of gut virome dysbiosis and RA or even gut virome dysbiosis and consequential changes in the gut microbiome with Prevotella copri becoming the dominant oral and gut bacteria. It is left for scientists to discover to what extent gut microbiome and gut virome follow independent trajectories or have interacting trajectories; if interacting, when and where are the points of interaction?

Prevotella and RA Pathogenesis
Most likely, P. copri enter the bloodstream and infiltrate joints via phagocytic cells (such as macrophages, neutrophils, and dendritic cells) [71]. P. copri infection causes massive production of immunogenic helper T cell (Th17) cells, proinflammatory cytokines (such as IL-6, IL-17, and IL-23), leading to inflammation in the gut and that subsequently migrates to other organs and initiates RA as shown in a preclinical study [69,72]. An increased number of P. copri in the large intestine causes inflammatory responses in the gut. Th17 cells, IL-17, bacterial DNA, and possibly Prevotella spp migrate to the joints and induce joint inflammation and RA [79][80][81] (Figure 1). Prevotella intermedia, (another Prevotella bacteria) has been shown to increase prostaglandin E2 from arachidonic acid by activating cyclooxygenase-2 in the joints [98,99]. Noticeably, overexpression of the cyclooxygenase-2 enzyme was observed in RA [100]. Increased prostaglandins have multiple roles in RA pathogenesis, such as pain in the joints, inflammation, bone metabolism, and immune response [99].
Prevotella is also present in the oral cavity, and their presence increases in periodontal diseases, and thus RA is also correlated with periodontitis [49,101,102]. Periodontitis is highly correlated with RA in the clinic, and the oral bacterial population shift towards Porphyromonas gingivalis and other anaerobic bacteria such as Prevotella spp. [91,103] (Table  1). A concomitant increase in Porphyromonas gingivalis in the oral cavity and P. copri in the intestine is observed in patients who experience RA [92] (Figure 1). Serum samples from patients with RA showed positive responses to immunoglobulin G (IgG) antibody of P. gingivalis [104]. P. gingivalis from the oral cavity possibly migrate to the synovial tissues through phagocytotic capture of dendritic cells, and P. gingivalis also causes cit-rullination of proteins in synovial tissues, which in turn, produce systemic inflammatory responses [91,93].
Besides these, P. histicola and P. intestinalis have shown immunomodulatory effects and reduced arthritis in mice similar to RA [82][83][84]. Prevotella intestinalis may cause a reduction in short-chain fatty acids and IL-18 production in the intestine, which reduces the abundance of acetate-producing bacteria like P. copri [82]. P. histicola protects the intestinal mucosal barrier by increased enzymatic expression of antimicrobial compounds. The bacteria also produce anti-inflammatory effects by reducing inflammatory responses of Th17 cells and increasing the number of regulatory T cells and IL-10 [83] (Table 1).

Roles of Lactobacillus Probiotics against RA in Preclinical Studies
Probiotics are live microorganisms intended to be administered orally to improve the host's gut microbiota [105,106]. Human gut microbiota sometimes becomes imbalanced and causes dysbiosis in situations like long-term treatment with antibiotics, NSAIDs, stress, and inflammatory conditions such as RA and osteoarthritis [107][108][109]. Bacteria like Lactobacillus spp. and Bifidobacterium spp. are widely used probiotics, and Lactobacillus spp can survive in an acidic gastrointestinal environment, and the presence of glucose in the gut help their survival [106,110]. The efficacy of probiotics depends on the microbial strain or pathophysiologic conditions of the host [109].
Treatment with L. casei or L. acidophilus over a period of 28 days reportedly prevented the development of arthritis in a preclinical model that reduced arthritic scores and proinflammatory cytokines such as IL-17, IL-1β, IL-6, and TNF-α, similar to that of indomethacin treatment [38]. The treatment with the Lactobacillus spp also increased the release of anti-inflammatory cytokines like IL-4 and IL-10 in the body fluids [38]. Consumption of L. acidophilus and L. casei reduces the oxidative stress of animals with collagen-induced arthritis [38]. Oral L. casei treatment also induced anti-inflammatory effects in rats in a collagen-induced arthritis model by inhibiting the COX-2 enzyme by reducing the proinflammatory cytokines [111]. Another preclinical study showed that intragastric administration of L. casei prevented the development of Salmonella enterocolitisinduced arthritis and reduced expression of proinflammatory cytokines (e.g., IL-1β, IL-6, IL-17, IL-23, and TNF-α) [85]. Treatment of L. casei at the initial stage of adjuvant-induced arthritis (AIA) model (similar to RA) in rats inhibited the development of arthritis that was comparable to methotrexate with normalization of gut microbiota and an increment of L. acidophilus population [86]. L. acidophilus treatment showed anti-inflammatory properties as it suppressed Th17 cell-mediated secretion of proinflammatory cytokines (IL-1β, IL-6, TNF-α, IL-17, and IL-23), but increased secretion of the anti-inflammatory cytokine (IL-10) [87,88]. Another study showed similar changes in the profile of cytokines along with reduced swelling, cartilage damage and lymphocyte infiltration in joints after L. casei treatment in collagen-induced arthritic rats [89].
On the negative side, L. casei [114], L. bifidus L. salivarius, L. iners, and L. ruminis are connected with pathogenesis of RA and increased populations of these bacteria have been observed in RA patients in comparison to healthy people [78]. Another study also showed that oral supplementation of L.casei and L. acidophilus increased phagocytic and lymphocytic activity in the intestinal mucosa of Swiss albino mice [115]. Therefore, the consumption of L.casei and L. acidophilus can sometimes but rarely produce harmful effects, which is probably due to the increased population of these bacteria in the human gut. There are variations of effects between Lactobacillus spp., treatment protocols and induction of experimental rheumatoid arthritis, animal species, and measurement parameters as reported previously [78]. Therefore, the outcome of these studies is quite inconclusive, but we hypothesize that a moderate population of L. casei and L. acidophilus produces beneficial effects. However, an increased population may cause infiltration of these bacteria from the gut to other organs, thereby producing harmful effects.

Roles of Lactobacillus Probiotics against RA in the Clinic
Patients with RA showed an increased presence of Bacteroides, Escherichia, and Shigella bacteria in the gut with a marked decrease in Lactobacillus spp. [116]. A well-balanced gut microflora provides essential vitamins like B-vitamins B3, B5, B6 (pyridoxal phosphate), B7, and B12, folate, tetrahydrofolate, and vitamin-K [58]. Low plasma vitamin-B6 has been observed in inflammatory conditions like RA, and long-term treatment with NSAIDs such as cyclooxygenase inhibitors prevented vitamin-B6 metabolism and thus reduced pyridoxal phosphate concentration from blood [117]. Impaired secretion of these essential vitamins (in conditions like gut dysbiosis), especially vitamin-B6 deficiency, can cause RA and cardiovascular complications [118,119]. Adequate colonization of Lactobacillus spp can improve the epithelium's integrity, and it becomes less susceptible to Bacteroides, Escherichia, and Shigella infections and their translocation into the intestinal lumen [120][121][122][123]. The bacteria secrete multiple short-chain fatty acids (e.g., lactic, acetic, and polyglutamic acid) and vitamins that provide nutritional support and decrease the pH of the intestinal lumen that prevent the colonization of harmful bacteria [124][125][126].
L. casei treatment over a period of 8 weeks improved RA-related pathophysiological parameters in a randomized clinical trial [127]. Lactobacillus spp also acts as an antimicrobial agent against different microorganisms [124,128]. Patients with RA show increased nitric oxide and reactive oxygenated species in blood and synovium, which cause degradation of lipids, other macromolecules, and matrix of the affected person [129,130].
Once daily administration of L. casei in capsule (contained 10 8 colony-forming unit) consumption treatment over a period of 8 weeks reduced the swelling of joints, arthritisrelated disease activity score-28, serum high-sensitivity C-reactive protein (hs-CRP) levels, and proinflammatory cytokines, especially TNF-α and IL-12 in women with RA relative to the placebo-treated control group. L. casei increased plasma IL-10 levels after the treatment but caused no changes in IL-1β and IL-6 levels [131]. Similarly, another study with a similar L. casei treatment protocol on a smaller number of patients found similar changes in the cytokines profile of RA patients [132]. The same dose of L. casei administered over eight weeks did not change the oxidative stress indicators or lipid profiles of RA patients [133,134]. Regular consumption of L. casei preserves the gastrointestinal diversity and prevents gastrointestinal dysbiosis, physiological stress, RA, and other inflammatory disorders [107]. L. acidophilus supplements in diabetic patients (n = 48, p= 24, c = 24) over a period of 4 weeks did not modify systemic inflammatory responses (induced by Escherichia coli lipopolysaccharide injections) and insulin sensitivity [135] ( Table 2). The randomized clinical trials of probiotics are not readily comparable among these, as there are variations in the patient selection, probiotic formulation, experimental parameters, dose, and frequencies of probiotic treatment, and all these can potentially affect the experimental outcomes (Table 2). RCT, 54 (C: 27, P:27) P: L. acidophilus (2 × 10 9 cfu/g), L. casei (2 × 10 9 cfu/g), B. bifidum (2 × 10 9 cfu/g), 0.

Possible Effector Molecules of Lactobacillus Species
Intestinal microbes and their gene products can interact with PRRs, including C-type lectin receptors, including the specific intracellular adhesion molecule-3 grabbing nonintegrin homolog-related 3 (SIGNR3) [140,141]. Any disruption of the delicate balance between the intestinal biome and their various gene product interactions can lead to disruptions in signal-transducing, giving rise to diseases like irritable bowel syndrome [142]. Lactobacillus acidophilus NCFM has been described as a model probiotic strain [143]. The strain is distinguished by its several surface (S) layer proteins. The S layer is encoded by three Slp-encoding genes: slpA (LBA0169), slpB (LBA0175), and slpX (LBA0512). SlpA, a probiotic factor, can bind to the host immune receptor SIGNR3, a C-type lectin. The binding affects modulatory signals, resulting in the mitigation of colitis, maintenance of a healthy gut biome, and protection of gut mucosal barrier function [143]. Other documented effector molecules (besides SlpA) in various species of Lactobacillus and Bifidobacterium strains have been reviewed by Lebeer and colleagues [143]. These molecules include specific pili (Lactobacillus rhamnosus GG), S-layer proteins (Lactobacillus acidophilus NCFM), exopolysaccharides and muropeptides. Various metabolites like tryptophan-and histaminerelated metabolites (Lactobacillus reuteri 6475), CpG-rich DNA, and enzymes like lactase and bile salt hydrolases can also act as effector molecules.
Lactobacillus, often neglected because of the notion that they are transient passengers of the gut [144], now are regarded as important members of the gut microbiome, because they can form up to 5% of the gut microbiota and up to 99% of vaginal microbiota [145]. However, not much study is available on the effector molecule(s) of Lactobaccilus casei and Lactobacillus acidophilus that may play a significant role in the attenuation of RA. Consumption of Lactobacillus casei fermented milk reportedly prevented Salmonella enteritidis reactive arthritis [85]. Such prevention occurred through modulation of IL23/IL17 expression. On the other hand, adhesion of probiotic bacteria to the gastrointestinal surface is considered a prerequisite for the exclusion of competitive pathogens [146], which would suggest that Lactobacillus casei adhesion molecules like fucose may play the part of an effector molecule [85].

Conclusions
In summary, preclinical studies mainly focus on the preventative roles of Lactobacillus spp in various experimental models of RA. In contrast, several clinical studies have investigated this bacteria's role in the improvement of the symptoms and diagnostic parameters after RA establishment. L. casei or L. acidophilus supplementation (in the form of food, drink, or pharmaceutical dosage forms) may not have an immediate favorable impact against RA. It ameliorates gut dysbiosis and consequential RA pathogenesis after repeated long-term use. However, an increased population of Lactobacillus salivarius was recorded in the gut, teeth and saliva of RA patients and L. salivarius and some other Lactobacillus spp. could be associated with RA pathogenesis [145,147]. Long-term high-dose consumption of some Lactobacillus spp. may induce rare complications like liver abscess and bacteremia [148][149][150]. Therefore, the benefits of Lactobacillus probiotic supplements depend on the Lactobacillus species. Since L. casei and L. acidophilus have anti-inflammatory, antimicrobial, antioxidant properties, these bacteria act symbiotically in the gut to establish their colonization and thus increase the integrity of cellular layers (of the gastrointestinal tract), maintain nutritional support of the host, and reduce the severity of inflammatory conditions like RA.
It has been put forward that the therapeutic use of probiotics and prebiotics in the case of obesity-related non-alcoholic fatty liver disease did not demonstrate any major benefits in high-quality clinical trial studies [151]. A number of factors are involved in probiotic studies, namely the initial composition of the patient's gut and possibly oral microbiome, the nature of probiotic administered, the progress of the disease at the time of probiotic administration, and the continuation of the diet and the probiotic along with any other medications. Every disease possibly has its own probiotics regimen and the initial microbiome of the patient, which brings in not one but two factors. As such, probiotics can be and more possibly should be regarded as nutraceutical supplements, and it will be somewhat futile to presume that what will not work for obesity-related non-alcoholic fatty liver disease will also not work for, say RA.
Probiotics can be used as nutritional supplements to manage inflammatory disorders like rheumatoid arthritis, diarrhea, dysentery, irritable bowel syndrome, diabetes, but the effectiveness in clinical trials is yet to be proved. Probiotic supplements should be considered as a functional food which has some therapeutic benefits. The quality of probiotic products is currently examined based on the number of viable colonies of certain probiotic bacteria (e.g., L. acidophilus) in the product [152]. However, the number of colonies cannot represent the effectiveness of the bacteria for inflammatory disorders like RA. Physical parameters, such as stability in a low pH environment (e.g., gastric lumen), physical stability, viability and proper storage condition, should be monitored to maintain good quality products [153]. In vitro laboratory assessments like adhesion capabilities of probiotic bacteria to human intestinal epithelial cells (such as Caco-2 cell line) and the capability to contribute to the lubrication of inner gut walls (in vivo) needs to be taken into account [152,154,155]. New quality control parameters should be implemented to maintain an optimum standard of probiotic products for consumers.