Traditionally recognized as a human pathogen, Helicobacter pylori (Hp)is a gram-negative microaerophilic bacterium that colonizes the human gut in early childhood and persists most often for life (1). Infection may be acquired through oral-oral or faecal- oral transmission. Improving hygienic conditions and socio economic status has reduced Hp status in developed/industrialised nations. This declining trend was noted to be associated with an increase in autoimmune disorders in the developed world (2, 3) and supports the “hygiene hypothesis”. The latter refers to the inverse relationship between infection and atopy that was first proposed by Strachan (4) in 1989. He observed an increased frequency of allergic rhinitis and atopic dermatitis among first born children who were less likely to have been exposed to infection compared to younger siblings. The term “hygiene hypothesis” coined in 2000 (5), laid emphasis on the broader environmental infection burden that showed a negative correlation between overall infection frequency and a substantial increase in frequency of allergic and autoimmune diseases, observed in industrialized countries. A variety of pathogens, parasites and commensal microorganism have been observed to protect against different autoimmune conditions and includes Hp (6).

Helicobacter pylori infection in childhood possibly contributes to the development of the immune system and may be protective against later onset of some autoimmune diseases such as atopy, allergy and MS. Despite high prevalence in the developing world, < 10% develop a chronic inflammatory state which leads to symptomatic gastroduodenal disease later in the life of the human host (7). This phase of hp infection is also associated with extra-gastric diseases including certain autoimmune disorders such as rheumatoid arthritis, inflammatory bowel disease (8) and NMOSD (9, 10). Several studies have reported the protective role of Hp in MS, including from Japan (9, 10,) Iran (11), India (12) and Australia (13). There were two studies that failed to show this protective effect, one of which had included patients with opticospinal MS along with conventional MS (14). The other study had a poorly designed control arm (15, 16). Recently 2 meta analysis which included several new studies also concurred with the protective role of hp in MS (17, 18). As early as 2007, there were reports of a higher frequency of Hp among opticospinal form of MS (which in all probability was NMOSD) when compared to conventional MS patients (9, 14) This was confirmed by one of the authors (14) in a later study that incorporated AQP4-IgG assay that showed an association with NMOSD but not MS.

Myelin oligodendrocyte glycoprotein antibody associated disorders (MOGAD) have been recently discovered. Antibodies targeting MOG which is a surface expressed protein in myelin results in demyelination similar to MS (19) with which it also shares pathological similarity (20). The association of Hp with MOGAD has not been reported before. The present study explores the frequency of Hp in all three primary autoimmune demyelinating CNS disorders in a developing country where there is high Hp seroprevalence, to determine its possible association.

Four hundred and fifty two patients, which included all 99 myelin oligodendrocyte glycoprotein antibody associated disorder (MOGAD) (21), all 99 aquaporin-4 antibody positive (AQP4 IgG+) NMOSD (22) and 254 consecutive patients with MS (23) from the Mangalore demyelinating disease registry [MANDDIR]were selected. Two hundred and forty three healthy volunteers matched by age and gender were included as controls for this study. Patient demographics, diagnosis, age at disease onset, duration and the last recorded expanded disability status scale (EDSS) was obtained from our data base. Socioeconomic and educational status (24) was queried using a previously validated questionnaire (12). Testing for serum Hp IgG was done using Vircell (Granada, Spain) ELISA kits as per manufacturer’s instructions. Antibody index was determined (by dividing optical density values of samples by optical density for cut-off control samples, multiplied by 10). Antibody index was positive if >11, equivocal if between 9 -11 and negative if < 9. All equivocal results were retested and if found to remain equivocal reported as negative for Hp IgG. All patients were tested for both AQP4 IgG (25) and MOG IgG using “in house” cell based assays. This study was approved by the institutional ethics committee and patients and healthy volunteers signed an informed consent form.

Clinical and Demographic features are listed in Table 1 . Patients in the MS group were predominantly of the relapsing remitting (RR). The non MS group comprised of equal number of AQP4IgG + NMOSD and MOGAD patients. Patients were predominantly female among MS and NMOSD patients. All patients had comparable age and disease duration at time of Hp serology testing.

Helicobacter pylori prevalence remains high in developing nations and offers an opportunity to study its association with autoimmune demyelinating CNS disorders in these regions. This is particularly relevant for Indian MS patients in whom there were no definitive environmental associations detected that were traditionally associated among white populations. These include Epstein Barr virus infection (26), smoking (12), obesity (12) and Vitamin D deficiency (27). In an earlier study we reported that Hp infection was protective against MS patients (12) and we have currently replicated the results in a larger cohort. Seropositive patients with MS- MOGAD had a later age of disease onset in our study. A similar delay in disease onset was reported in Hp seropositive patients with bronchial asthma (28). Despite a longer duration of disease, seropositive patients had disability that was comparable with seronegative patients ( Supplementary Table 2 ). Previously two case control studies have reported similar results in Hp seropositive MS patients (9, 11). Our study also suggests that Hp infection may confer a protective effect against MOGAD similar to MS. In contrast, Hp infection was detected to be frequent in NMOSD patients. An effect on disease onset and course could not be clearly determined in the latter.

The differential association of CNS autoimmune disorders with Hp infection may in part be due to the differential immune response mounted by Hp in the human host (29). It is evident from human gut mucosal studies that Hp behaves as a commensal in early infection with a potential for pathogenicity in later life. Compared to adults, gastric mucosa in paediatric patients showed minimal inflammation, increased T regulatory (reg) cells and elevated levels of Treg related cytokines namely transforming growth factor- beta (TGFβ) and interleukin 10 (IL10) (30). Limited studies in experimental allergic encephalitis (EAE) models demonstrated that prior infection with Hp induced Foxp3⁺Treg cell upregulation (31) and inhibition of MOG specific Th1 and Th17 responses (32). In adult patients, peptic ulceration was noted to be associated with reduced Tregs (33).

We have hypothesised possible mechanisms by which Hp infection could modulate the host immune response. Recent insights (34) suggest that Hp may possess inflammasone ligands that trigger caspase- 1 activation in dendritic cells lining the gastric lumen ( Figure 1 ). Activation of the Toll like receptor2/Nucleotide-Binding Domain, Leucine-Rich–Containing Family, Pyrin Domain–Containing-3/Caspace1/Interleukin-18 axis (TLR2/NLRP3/CASP1/IL-18) axis may promote IL18 mediated Treg upregulation in the early phase of infection. The resulting immune tolerance may be the basis for protection against allergy and possibly autoimmune disorders such as MS and MOGAD. On the other hand, chronic gastritis is characterized by upregulated T helper 17(Th17) response, neutrophil recruitment and expression of B cell activating factor (BAFF) in macrophages (35, 36). Chronic Hp infection has also been noted to be associated with gut dysbiosis and associated pro inflammatory state (37). We have postulated that pro- inflammatory state of chronic gastritis accompanied by gut dysbiosis may upregulate Th1/Th17 driven immune response initially in the gut and later systemically, facilitating the development of NMOSD in later life. The role of gastric derived antibodies that cross react with neural elements cannot be excluded. A previous publication from our group has shown this to be a potential pathway for disease pathogenesis in NMOSD (38). Helicobacter pylori related pro-inflammatory proteins particularly neutrophil activating protein (NAP) may contribute to AQP4 –IgG associated neural damage and severity of disease.In a recent study a significant correlation was noted between seropositive AQP4 IgG + NMOSD, Hp- NAP and disability (10). Bacterial virulence genes of Hp have not been studied in detail in the context of CNS autoimmune disorders. An association between Hp- cytotoxin associated gene A (cag-A) and autoimmune thyroiditis (39) and asthma was previously reported (28). Additionally the underlying genetic susceptibility for autoimmune disorders is also important.

We reviewed the relevance of hygiene hypothesis in a low middle income (LMIC) setting in relation to Hp infection and CNS autoimmune disorders. For this purpose,we analysed educational status and income levels of patients and primary caregivers in childhood in order to correlate poor sanitary conditions in childhood with early Hp colonization in the gut. Our study showed a significant association between lower educational status among all cases and controls stratified by Hp serology. Our hospital caters to patients from lower economic status which may explain the lack of difference in economic status between the two groups.

In conclusion, in developing countries Hpylori may be a significant environmental factor associated with autoimmune demyelinating disorders. Our study was limited by the small number of study participants and the cross sectional nature of the analysis. It is not clear at this time whether analysis of risk/benefit has to be carefully undertaken before Hp eradication is contemplated, in populations harbouring high Hp seroprevalence. Therefor the role of H pylori in modulating human immunity and its protective/deleterious effects need to be understood through larger studies that will additionally evaluate the role of Hp virulence genes.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding author.

The studies involving human participants were reviewed and approved by Central Ethics Committe, Nitte University. The patients/participants provided their written informed consent to participate in this study.

LP developed the concept, study design, analysis, interpretation, drafting and revising the work. CM contributed to study design, data acquisition, analysis, interpretation of results, manuscript drafting and revision. AD’C contributed to data acquisition, analysis and manuscript drafting. AS contributed to data collection, data analysis and interpretation. All authors contributed to the article and approved the submitted version.