Proton pump inhibitors (PPIs), such as omeprazole, are among the top 10 most commonly prescribed drugs in the US (1), used for the treatment of acid-related digestive system diseases, such as esophagitis, gastroesophageal reflux disease, peptic ulcer disease among few. Due to the excellent safety profile of PPIs, long-term, even lifelong use is not uncommon (2). However, PPIs are one of the drugs with the strongest potential to modulate gut microbiota composition (3).

PPIs upon activation posed by acid-induced cleavage within acidic secretory canaliculi, inhibit H⁺/K⁺ ATPase of gastric parietal cells blocking acid production (1). Increased gastric pH impedes vitamin B12, iron, and magnesium absorption and alters intestinal microbiota composition (4, 5) by enabling bacteria translocation across the gastric barrier, changing digestive content and its distribution across the digestive system, influencing hormone balance (6), as well directly impacting bacterial H⁺/K⁺ ATPase of several microorganisms (7). PPIs application decreases the abundance of intestinal bacteria, simultaneously increasing the number of oral bacteria in the intestines (47). Although PPIs-induced gut microbiota alteration is implicated in increased susceptibility to Clostridium difficile–associated disease and promotes intestinal bacterial overgrowth (8) several studies showed anti-inflammatory properties of PPIs (9).

In vitro studies showed that PPIs inhibit reactive oxygen production by up-regulating SOD and catalase, reduce the production of proinflammatory TNF-α, IL-1β, and IL-6 cytokines (10, 11), and the production of Th2-type cytokines IL-4 and IL-13 (12). Furthermore, PPIs reduce neutrophil degranulation and chemotaxis (13), which could be a consequence of reduced expression of endothelial cell intercellular adhesion molecule-1 (ICAM-1) and vascular adhesion molecule-1 (VCAM-1) after PPIs treatment (14, 49). However, the anti-inflammatory properties of PPIs did not manage to ameliorate clinical scores in experimental autoimmune encephalomyelitis (EAE) (15), despite changes in gut microbiota profile (15). Contrary omeprazole administration effectively ameliorated ovalbumin-induced asthma, what may be related to reduce Th2-type cytokines production (12).

Contact hypersensitivity (CHS) is an animal model of allergic contact dermatitis (ACD), a disease that arises from exposure to low-molecular-weight substances and ions and affects up to 15% of the general population and 30% of working people (16). CHS is a type IV hypersensitivity reaction mediated by T cells, the mechanism that drives many autoimmune disorders (17).

CHS consists of inductive and effector phases. During induction, skin exposure to hapten solution results in self-protein modification by hapten and activation of innate immune response in the skin. Proinflammatory IL-1β and TNF-α activate dermal dendritic cells (DDC), which pick up haptenized-self protein and migrate to draining lymph nodes, where the hapten-specific CD4⁺ and CD8⁺ Th1 cells and Th17 cells are induced (18–20). The effector phase is induced after 4-5 days by applying a hapten solution somewhere else in the body. Migration of hapten-specific T cells to the site of subsequent exposure, along neutrophils and macrophages, driven by expression of adhesion molecules on endothelial membranes, results in skin inflammation (skin swelling) that peaks 24 to 48 h after hapten challenge.

The magnitude of CHS response is controlled during induction by the levels of proinflammatory IL-1β and TNF-α and anti-inflammatory IL-10 during immunization (18) and the proinflammatory potential of dendritic cells (50) dependent on the production of reactive oxygen species (ROS) and proinflammatory cytokines (21). Various populations of regulatory cells were implicated in the modulation of induction and elicitation of CHS (17), and their induction is influenced by gut microbiota composition (17).

Given the common long-term use of PPIs and their potential to modulate gut microbiota composition—a well-known factor in regulating systemic immune response—our study aimed to investigate the consequences of prolonged omeprazole use on the development of T cell-dependent immune responses.

The contact hypersensitivity response (CHS) is a mouse model of allergic contact dermatitis observed in humans, arising after exposure to contact allergens. These low-molecular-weight substances, also known as haptens, include chemicals widely used by society, such as metal ions, dyes, preservatives, and fragrances (23). An estimated 20% of the general population suffers from contact allergy (24). The CHS reaction is mediated by CD4⁺T cells (25) producing IL-17A and IFN-γ (19) and is classified as a type IV hypersensitivity reaction, which characterizes many autoimmune disorders.

Omeprazole is one of the most commonly prescribed drugs (1), and its use could be lifelong. It blocks gastric acid production, leading to changes in intestinal microbiota composition (48). Additionally, it shows anti-inflammatory properties as it regulates ROS and pro-inflammatory cytokine production and suppresses immune cell chemotaxis (10, 13). Since exposure to contact sensitizers and omeprazole is very common, and omeprazole presents immune modulatory properties, this study aimed to evaluate the impact of omeprazole treatment on the development of T cell-dependent immune response using the CHS model.

Previous studies showed that CHS reaction is influenced by a plethora of factors such as antibiotics (17), diet (51), antidepressant drugs (52), which also acts through modulation of intestinal microbiota. In this study, we evaluated the impact of omeprazole on the development of CHS response. Our data indicate that two-week treatment with omeprazole before induction of CHS response reduces the severity of inflammatory response measured by ear swelling ( Figure 1A ), which is confirmed by reduced ear biopsy weight ( Figure 1B ) (26).

Ear swelling 24 h after subsequent exposure to hapten drives infiltration of antigen-specific T lymphocytes and innate immune cells such as MPO-positive neutrophils (26). Our data shows that omeprazole treatment leads to reduced MPO activity in ear biopsies ( Figure 1C ). Additionally, reduced neutrophil infiltration could, in turn, diminish the magnitude of CHS response, as both neutrophils and MPO are implicated in CHS elicitation. Mice depleted of neutrophils transferred with CHS T-effector cells fail to elicit an ear swelling response (27), while MPO deficiency reduces vascular permeability (28).

Reduced CHS response was accompanied by reduced concentration of proinflammatory IL-6 and TNF-α in ear extracts ( Figure 1D ) and IL-6 in ELN cell culture ( Figure 1E ). Both IL-6 and TNF-α could be classified as innate immune cytokines, which are produced by keratinocytes directly stimulated with haptens, or by skin residing innate immune cells stimulated by danger associated molecular patterns (DAMPs) released after hapten application (29). Both cytokines positively regulate CHS response, as IL-6 knock-out and TNF-α knock-out mice have ameliorated ear swelling response (30, 31). Keratinocytes, Langerhans cells, macrophages, or innate lymphoid cells 1 (ILC1) are known sources of TNF-α, which is implicated in T cell infiltration during elicitation by induction of IFN-γ-inducible protein 10 (IP-10, CXCL10) (29, 30). Keratinocytes, Langerhans cells, and macrophages produce IL-6, supporting lymphocyte proliferation in lymph nodes (31).

After painting with hapten, skin resident Langerhans cells pick up antigens and migrate to local draining lymph nodes where the hapten-specific T cells are induced. CHS-mediating T cells belong to the CD8⁺ or CD4⁺ population and produce IL-17A or IFN-γ (19) as knock-out mice of IL-17A and IFN-γ (32), and mice deficient in receptors for IL-17A, or IFN-γ have reduced CHS response (19). Subcutaneous injection of recombinant IL-17A or IFN-γ enhanced CHS manifestation, while depletion using mAb of IFN-γ in IL-17 R^−/− mice and depletion of IL-17A in IFN-γ R^−/− mice further suppressed the CHS response compared to IL-17 R^−/− or IFN-γ R^−/− (19). Our data shows that omeprazole application prior to immunization results in a reduced percentage of TCRαβ⁺ CD4⁺ IFN-γ⁺ and TCRαβ⁺ CD4⁺ IL-17A⁺ lymphocytes in ALNC ( Figure 2A ) and TCRαβ⁺ CD4⁺ IL-17A⁺ and TCRαβ⁺ CD8⁺ IL-17A⁺ lymphocytes in SPLC ( Figure 2B ), suggesting that the application of omeprazole interferes with the induction of antigen-specific T cells. Furthermore, the development of CHS response is supported by γδ⁺T cells, as adoptive transfer of LN cells depleted of γδ⁺T reduces the ear swelling response (33, 48). The γδ⁺T cells are a known source of IL-17A, but could also promote the CHS response by supporting IFN-γ, IL-12, and TNF-α production by LN cells (33). In agreement with the role of γδ⁺T cells, we observed that reduced CHS response after omeprazole application is associated with a lower frequency of TCRγδ⁺ IL-17A⁺ lymphocytes in ALNC ( Figure 2A ).

Although the depletion studies revealed that CHS-effector T cells belong to CD8⁺ and CD4⁺ populations, they also showed that some fraction of CD4⁺ could have regulatory properties (25). Further research revealed that CHS response is negatively regulated by TCRαβ⁺ CD4⁺ CD25⁺ FoxP3⁺ Treg cells, CD19⁺ B220⁺ CD5⁺ IL-10⁺ Breg cells, IL-10⁺ Tr1, or IL-10⁺ TCR γδ⁺ lymhocytes (17, 34). FACS analysis of LN cells from omeprazole-treated mice showed an increased number of CD4⁺ CD25⁺ FoxP3⁺ Treg, and CD4⁺ IL-10⁺ Tr1 cells, suggesting that omeprazole application favors the development of T regulatory cells ( Figure 2C ). Consistent with our results, omeprazole treatment increases the frequency of CD4⁺ CD25⁺ FoxP3⁺ Treg cells and decreases the frequency of CD4⁺ IL-17A⁺ T cells among peripheral blood mononuclear cells (PBMCs) of patients with duodenal ulcers. The shift was accompanied by a reduced concentration of IL-17A and an increased concentration of IL-10 (35). Treg cells restrict inflammatory response by preventing the induction of antigen-specific cells and limiting their proliferation. Consistently, our data shows a reduced frequency of IFN-γ⁺- and IL-17A⁺ -producing T cells.

To further understand immune changes induced by omeprazole application, we evaluated the immune status in the intestine and gut-associated lymphoid organs. Previous studies showed that reduced CHS response due to enrofloxacin-induced dysbiosis was associated with the anti-inflammatory milieu in the intestinal tissue, characterized by increased production of IL-10 and no change in the level of IFN-γ and IL-17A (50). Similarly, a two-week omeprazole application leading to reduced CHS, increased the concentration of anti-inflammatory IL-10 and had no impact on pro-inflammatory IL-17A and IFN-γ concentrations in small intestine biopsies ( Figure 3A ). The increase in IL-10 production after omeprazole application is consistent with study showing that patients with gastric ulcer treated with omeprazole combined with antibiotic mixture have increased levels of IL-10 in serum and in the ulcer tissue compared to patients treated with bismuth subcitrate combined with antibiotic mixture (35). IL-10 was implicated in the suppression of ROS production pro-inflammatory IL-1β, IL-6, and TNF-α, and suppression of adaptive immune response induction by inhibiting antigen presentation through the reduction of MHC II and costimulatory molecule expression (50). Consistently, we observed reduced production of IL-6 and TNF-α by MLN cells ( Figure 3B ). Additionally, the reduced production of IL-6 and TNF-α could also be the consequence of the direct influence of omeprazole on immune cells, as the studies evaluating the impact of PPIs on immune response, showed decreased production of TNF-α and tendency towards reduced production of IL-6 by THP-1 cell line exposed to Lansoprazole (10). However, another study reported that two-weeks omeprazole application restored to the control level the production of IL-1β, IL-6, and TNF-α in nerve tissue homogenate from chronic constriction injury (CCI)-induced rat model of neuropathic pain (11).

The cellular sources of IL-6 and TNF-α are dendritic cells and macrophages (29). In our study, we also observed that omeprazole application reduced the frequency of DC CD11c⁺ MHCII⁺, migratory DC CD11c⁺ MHCII⁺ CD103⁺ ( Figure 3C ), and CD11b⁺ Ly6C⁺ monocytes/macrophages ( Figure 3E ). Both cytokines play a crucial role in the development of Th17 cells, as IL-6 prime the development of Th17 cells while the influence of TNF-α is indirect, as it promotes production of IL-1β and IL-6 from monocytes (36). CD11c⁺ MHCII+ CD103⁺ DCs are the principal fraction of migratory DC in MLNC and are known sources of IL-6 required for induction of Th17-dependent immune response (37). Additionally, CD103+ DCs, but not CD103− DCs, support the induction of CD4+CD25+FoxP3+ Treg cells. Mice lacking CD103+ antigen-presenting cells were unable to restrict T cell-mediated colitis (38, 39). The migration of CD11c+ MHCII+ CD103+ DCs to the MLN is driven by chemokines (40), which are also produced by the lymph nodes and spleen, suggesting the potential for CD103+ DCs to migrate to peripheral tissues, where they could support the induction of Treg cells (41).

Additionally, we found that omeprazole application reduced the production of ROS, as we observed reduced frequency of CD11c⁺ MHCII⁺ ROS⁺ DC ( Figure 3C ) and CD11b⁺ Ly6C⁺ROS⁺ monocytes/macrophages ( Figure 3E ). This is consistent with the studies showing that omeprazole application increase the level of antioxidant superoxide dismutase (SOD), and concomitantly the level of reduced glutathione (GSH) (11). ROS produced by DC supports the induction of adaptive immune response, and inhibition of redox-sensitive pathways prevents the induction of antigen-specific T cells in the CHS model (21). Thus, reduced level of ROS, along with reduced frequency of CD11c⁺ MHCII⁺ CD103⁺ DC and lower production of IL-6 and TNF-α after omeprazole treatment, could account for impaired induction of TCRαβ⁺ CD4⁺ IL-17A⁺ and TCRαβ⁺ CD8⁺ IL-17A⁺ T cells ( Figure 2A and Figure 2B ), leading to reduced CHS response.

Dysbiosis is a change in bacterial composition, due to loss of beneficial bacteria, overgrowth of potentially pathogenic ones, or decrease in bacterial diversity or distribution within intestines (42). The application of PPIs changes the composition of intestinal microbiota (47). Gut microbiota is an established modulator of peripheral immune response, including the CHS model (17). Our data shows that a two-week omeprazole treatment changes intestinal microbiota composition. However, we did not observe changes in the relative abundance of Enterococcus spp., Bacteroidetes ( Figure 4B ), as well Clostridium coccoides (cluster XIVa), Actinobacteria and Clostridium cluster I ( Figure 4C ); omeprazole application increases the abundance of Lactobacillus, Clostridium coccoides – E. rectale (cluster XIVab) and SFB ( Figure 4A ). Although the impact of omeprazole on gut microbiota is undisputable, the exact changes could vary depending on dosing regimen, and tested model (15, 48).

Consistently to our study, a 4-week omeprazole treatment caused a major increase in Lactobacillus levels (43). Lactobacillus strains are well-known probiotics with proven anti-inflammatory action. Supplementation with Lactobacillus johnsonii efficiently reduced DSS-induced colitis, which was associated with decreased levels of TNF-α and IL-6 and increased levels of IL-10 and IL-17 (44). Additionally, bacteria belonging to Clostridium cluster XIVa support induction of FoxP3-expressing T cells (45), and the increase in their abundance after antibiotic treatment was associated with reduced CHS response (17). The role of omeprazole-induced dysbiosis in the regulation of immune response in the CHS model was confirmed using FMT ( Figure 4D ). The decreased ear swelling response correlated with a reduction in ear biopsy weight ( Figure 4E ), MPO activity ( Figure 4F ), and the concentrations of IL-6 and TNF-α ( Figure 4G ). These changes were observed following a two-week omeprazole treatment administered prior to immunization and challenge ( Figures 1A–D ). Additionally, flow cytometry analysis of ELN revealed an increased frequency of TCRαβ+ CD25+ FoxP3+ regulatory cells ( Figure 4H ), which, contribute to maintaining tolerance (46). Our data suggest that at least partially that the ameliorated CHS response after omeprazole treatment is the consequence of gut microbiota changes.

In conclusion, a two-week omeprazole treatment reduces CHS response by shifting immune status towards anti-inflammatory, characterized by increased IL-10 and decreased IL-6 and TNF-α production, which could be the consequence of the direct impact of omeprazole on immune cells or alteration of gut microbiota composition. The changes in immune cells and microbiota profiles after omeprazole application support the development of Treg cells while preventing the induction of Th17 lymphocytes. Alteration of Th17/Treg cells balance observed after omeprazole treatment suppresses CHS response.