Pseudomonas aeruginosa (Psae) constitute Gram-negative, non-fermenting bacteria that grow under strictly aerobic conditions and can be preferably found in moist environments (1). Bacterial motility and adhesive properties are provided by a single flagellum and several cell surface pili. A plethora of additional virulence factors such as alginate secretion, biofilm formation, quorum-sensing, an elaborate secretion system as well as intrinsic expression of antibiotic resistance genes facilitate adaptation to the respective environment as well as immune escape and establishment in the ecosystem of the vertebrate host (1, 2). Psae are considered opportunistic pathogens, but may cause urinary tract infections, ventilator-associated pneumonia, surgical and burn wound as well as bloodstream infections, particularly in hospitalized individuals with immunosuppressive comorbidities such as neutropenia that are associated with mortality rates of >30% (3). Furthermore, patients suffering from chronic respiratory morbidities including chronic obstructive lung disease, bronchiectasis, or cystic fibrosis are prone to chronic Psae infection, further exacerbating the underlying disease (3). Particularly the emergence of multidrugresistant (MDR) Psae strains due to bacterial expression of resistance genes such as β-lactamases, 16S rRNA methylases, and carbapenemases in recent years leading to severe infections with high mortality rates due to very limited treatment options has prompted the World Health Organization (WHO) in 2017 to rate MDR Gram-negative bacteria including Psae as serious global threats to human health, further emphasizing the need for novel (antibiotics-independent) treatment strategies (4).

In the healthcare setting, contaminated devices including respiratory equipment, but also water bottles and sinks are typical external sources for Psae acquisition (5, 6). Whereas, Psae is not considered as part of the commensal gut microbiota, a previous report revealed that rectal opportunistic bacterial carriage was a predictor for Psae-associated infections in patients admitted to an intensive care unit (ICU) lateron (7). Thus, the human gastrointestinal tract (GIT) should be taken into consideration as a potential internal Psae reservoir. Particularly in cases, when the integrity of the complex gut microbiota is disturbed following antimicrobial therapy, for instance, the compromised colonization resistance [physiologically preventing the host from establishment of (opportunistic) pathogens (8, 9)] may facilitate stable Psae colonization within the human gastrointestinal ecosystem (10, 11). In an earlier report, Psae could be isolated from fecal samples of healthy volunteers up to 1 week following ingestion with decreasing loads over time, whereas the challenged individuals did not exhibit any clinical complications (12). Valid information regarding the immunopathological potential of intestinal Psae colonization in the vertebrate host is scarce, however.

Recently, our group provided evidence for the first time that intestinal carriage of a clinical MDR Psae isolate resulted in pronounced pro-inflammatory sequelae in intestinal as well as systemic compartments of healthy wildtype (WT) mice (13, 14). Furthermore, pre-existing inflammatory conditions in the gut facilitated stable intestinal Psae colonization in mice (15, 16), which subsequently aggravated chronic colitis (16). In the present study we addressed whether peroral challenge of mice suffering from more acute intestinal inflammatory conditions in a different anatomic compartment within the GIT (namely, subacute ileitis) worsened the outcome of the underlying disease. Since the host specific gut microbiota is known to be essentially involved in the onset, progress and outcome of distinct immunopathological conditions including intestinal inflammation (8, 9, 17, 18), we aimed at mimicking human gut microbiota conditions in the applied experimental model. We therefore generated mice that were harboring a human gut microbiota following human fecal microbiota transplantation (FMT) and induced subacute ileitis by peroral low-dose Toxoplasma gondii infection as described by us recently (19, 20). We here show that approximately one third of human microbiota associated (hma) mice with subacute ileitis harbored Psae in their intestines upon oral challenge. The opportunistic pathogen was, however, responsible for pro-inflammatory immune responses in intestinal, extra-intestinal and even systemic compartments and, thus, worsened subacute ileitis outcome irrespective of the Psae-carrier status.

**Abbreviations:** CFU, colony forming units; FMT, fecal microbiota transplantation; GIT, gastrointestinal tract; H&E, hematoxylin and eosin; hma, human microbiota associated; HPF, high power field; ICU, intensive care unit; MDR, multidrugresistant; MLN, mesenteric lymph nodes; NO, nitric oxide; PBS, phosphate buffered saline; Psae, Pseudomonas aeruginosa; SPF, specific pathogen free; TLR, Toll-like receptor; Treg, regulatory T cells; WHO, World Health Organization; WT, wildtype

#### MATERIALS AND METHODS

#### Ethics Statement

Mouse experiments were performed in accordance of the European Guidelines for animal welfare (2010/63/EU) and had been approved by the commission for animal experiments headed by the "Landesamt für Gesundheit und Soziales" (LaGeSo, Berlin; registration numbers G0097/12 and G0039/15) before. Clinical conditions including body weight loss were assessed daily to assure animal welfare. Mice displaying over 20% weight loss were subjected to cervical dislocation in accordance with the guidelines of the local legal authorities.

#### Generation of Mice With a Human Gut Microbiota by Fecal Microbiota Transplantation

Female C57BL/6j mice were maintained under specific pathogenfree (SPF) conditions in the Forschungseinrichtungen für Experimentelle Medizin (FEM, Charité - University Medicine, Berlin, Germany). Microbiota depleted (i.e., secondary abiotic) mice were obtained following broad-spectrum antibiotic treatment as described earlier (8, 17). In brief, 8-week-old mice were treated with a quintuple antibiotic cocktail containing ampicillin plus sulbactam (1 g/L; Ratiopharm, Germany), vancomycin (500 mg/L; Cell Pharm, Germany), ciprofloxacin (200 mg/L; Bayer Vital, Germany), imipenem (250 mg/L; MSD, Germany), and metronidazole (1 g/L; Fresenius, Germany) via the drinking water for 8 weeks (ad libitum). Both, culture and 16S rRNA based molecular methods revealed virtual absence of bacteria in fecal samples as described previously (8, 18). Three days before human FMT the antibiotic cocktail was replaced by sterile tap water (ad libitum) to assure antibiotic washout (**Supplemental Figure S1**). Fresh fecal samples free of enteropathogenic bacteria, viruses, and parasites were collected from five individual healthy human volunteers, dissolved in sterile phosphate buffered saline (PBS; Gibco, Life Technologies, United Kingdom) and stored at −80◦C as stated elsewhere (8). Individual fecal aliquots were thawed immediately before FMT and pooled. In turn, secondary abiotic mice were perorally challenged with the human fecal donor suspension on three consecutive days by gavage (8). Notably, the inter-experimental variations in bacterial loads of the donor suspensions were < 0.5 logarithmic orders of magnitude (**Supplementary Figure S2**). In order to guarantee proper establishment of the complex human gut microbiota within the murine host, mice were kept for 10 days before subacute ileitis induction. Immediately before peroral T. gondii infection (day 0) individual fecal samples were collected for quantitative molecular analyses of main intestinal bacterial communities as described elsewhere (8, 17, 21).

### Molecular Analysis of the Human Fecal Donor Suspensions and the Intestinal Microbiota

Fresh ileal and colonic luminal samples were immediately snap-frozen in liquid nitrogen and stored at −80◦C until further processing. DNA was extracted from fecal samples as reported earlier (17). In brief, for quantification of DNA the Quant-iT PicoGreen reagent (Invitrogen, UK) was applied and DNA in samples adjusted to 1 ng per µL. Then, total eubacterial loads as well as the main bacterial groups within the human gut microbiota such as enterobacteria, enterococci, lactobacilli, bifidobacteria, Bacteroides/Prevotella species, Clostridium coccoides group, and Clostridium leptum group were surveyed by quantitative real-time polymerase chain reaction using genera-, group- or species-specific 16S rRNA gene primers (Tib MolBiol, Germany) as stated elsewhere (21, 22) and numbers of 16S rRNA gene copies per ng DNA of each sample assessed.

#### Induction of Subacute Ileitis

On day 0, mice were infected perorally with one cyst of T. gondii ME49 strain (in a volume of 0.3 mL brain suspension) by gavage in order to induce subacute ileitis (**Supplemental Figure S1**), as reported recently (19).

# MDR *P. aeruginosa* Infection and Quantitative Assessment of Fecal Bacterial Loads

The MDR Psae isolate was initially isolated from respiratory material of a patient with nosocomial pneumonia and kindly provided by Prof. Dr. Bastian Opitz (Charité–University Medicine, Berlin, Germany). Notably, the bacterial strain displayed antimicrobial sensitivity to fosfomycin and colistin only (15). Prior infection, the Psae strain was grown on cetrimide agar (Oxoid) for 48 h in an aerobic atmosphere at 37◦C.

On day 5 post ileitis induction, mice were perorally challenged with 109 colony forming units (CFU) of the MDR Psae strain by gavage in a total volume of 0.3 mL PBS as reported earlier (**Supplemental Figure S1**) (15).

For quantitative assessment of Psae colonization densities in the intestinal tract post-challenge, fecal and intestinal luminal samples were homogenized in sterile PBS. Serial dilutions were then streaked onto Columbia agar supplemented with 5% sheep blood (Oxoid, Germany) and onto cetrimide agar and incubated in an aerobic atmosphere at 37◦C for 48 h as described previously (15). Difference of the sample weights before and after asservation revealed respective fecal weights. The detection limit of cultivable bacteria was ∼100 CFU per g.

# Cultural Survey of Bacterial Translocation

In order to survey viable bacteria translocating from the intestinal tract to extra-intestinal and systemic compartments, ex vivo biopsies derived from mesenteric lymph nodes (MLN), spleen, liver, kidney and lung were homogenized in sterile PBS and analyzed in serial dilutions on respective solid media as described earlier (17, 23, 24). Cardiac blood was incubated in thioglycolate enrichment broths (BD Bioscience, Germany) for 1 week at 37◦C and streaked onto solid media thereafter (15). Bacteria were grown at 37◦C for at least two and t3 days under aerobic, microaerobic and anaerobic conditions.

#### Clinical Conditions

Macroscopic and / or microscopic occurrence of blood in murine fecal pellets was surveyed in individual mice on a daily basis by the Guajac method (Haemoccult; Beckman Coulter/ PCD, Germany) as stated elsewhere (25).

#### Sampling Procedures

96 h following Psae challenge (i.e., day 9 post ileitis induction), mice were sacrificed by isoflurane treatment [Abbott, Germany (**Figure S1**)]. Cardiac blood (for serum) and tissue samples from spleen, liver, lung, heart, MLN, ileum, and colon were removed under sterile conditions. Respective ex vivo biopsies were collected from each mouse in parallel for microbiological, immunological and immunohistochemical analyses.

#### Histopathology

Ex vivo biopsies taken from the terminal ileum were fixed in 5% formalin and embedded in paraffin. Sections (5µm) were stained with hematoxylin and eosin (H&E) and subjected to a standardized histopathological scoring system ranging from 0 to 6 as described in more detail earlier (17).

#### Immunohistochemistry

Five µm thin paraffin sections of ileal and colonic ex vivo biopsies were used for in situ immunohistochemical analysis as reported previously (21, 26–28). In brief, to assess apoptotic cells, proliferating cells, T lymphocytes and regulatory T cells (Treg), primary antibodies against cleaved caspase-3 (Asp175, #9661, Cell Signaling, Leiden, Netherlands; 1:200), Ki67 (clone 16A8, #652401, BioLegend/Biozol, Eching, Germany; 1:200), CD3 (#IR50361-2, Dako, Santa Clara, CA, USA; 1:5) and FOXP3 (clone FJK-165, #14-5773, eBioscience, Frankfurt, Germany; 1:100), respectively, were used. An independent blinded investigator determined the average numbers of positively stained cells in at least six high power fields (HPF, 0.287 mm2 ; 400 x magnification).

### Cytokine Detection

Ex vivo biopsies (∼1 cm2 ) derived from colon and ileum (both cut longitudinally and washed in PBS), as well as from liver (∼1 cm2 ), kidney (cut longitudinally, one half), lung (one organ), spleen (one half), and MLN (3 single lymph nodes) were transferred to 24-flat-bottom well culture plates (Falcon, Germany) containing 500µL serum-free RPMI 1640 medium (Gibco, life technologies) supplemented with penicillin (100 U/mL, Biochrom, Germany) and streptomycin (100µg/mL; Biochrom). After 18 h at 37◦C, IFN-γ, TNF, MCP-1, IL-6, and IL-10 concentrations were measured in culture supernatants and serum by the Mouse Inflammation Cytometric Bead Assay (CBA; BD Bioscience) applying a BD FACSCanto II flow cytometer (BD Bioscience). Nitric oxide (NO) concentrations were determined by the Griess reaction as described earlier (17).

# Statistical Analysis

Medians and levels of significance were determined by the oneway ANOVA test followed by Tukey post-correction test for multiple comparisons. Two-sided probability (p) values ≤0.05 were considered significant. Experiments were reproduced three times.

FIGURE 1 | Intestinal MDR Pseudomoas aeruginosa loads following peroral challenge of mice with a human gut microbiota suffering from subacute ileitis. Secondary abiotic mice were generated by broad-spectrum antibiotic treatment and associated with a complex human gut microbiota by fecal microbiota transplantation. Ten days thereafter, subacute ileitis was induced by peroral infection of human microbiota associated mice with one cyst of T. gondii ME49 strain (day 0). On day 5 post ileitis induction (p.i.), mice were perorally challenged with MDR P. aeruginosa (Psae) and (A) the fecal Psae loads were followed up for further 96 h by culture. (B) In addition, Psae counts were assessed in the small as well as large intestinal lumen 96 h post Psae challenge. Box plots represent the 75th and 25th percentiles of the median (black bar inside the boxes). Total range and significance levels determined by one-way ANOVA test followed by Tukey post-correction test for multiple comparisons are shown. Numbers of mice harboring Psae in the respective intestinal compartment out of the total numbers of analyzed animals are given in parentheses. Data were pooled from four independent experiments.

# RESULTS

# Colonization Properties of MDR *P. aeruginosa* in the Intestinal Tract of Mice With a Human Gut Microbiota Suffering From Subacute Ileitis

In the present study we raised the question whether subacute ileitis predisposed mice with a human gut microbiota for intestinal MDR Psae carriage and whether intestinal, extraintestinal or even systemic inflammatory sequelae might be induced following peroral Psae challenge. To address this, secondary abiotic mice were associated with a complex human gut microbiota by FMT on three consecutive days. Ten days later (i.e., on day 0), subacute ileitis was induced by peroral low-dose T. gondii infection of hma mice. On day 5 post ileitis induction, mice were perorally challenged with 109 CFU of a clinical MDR P. aeruginosa isolate by gavage and the fecal opportunistic pathogenic loads surveyed thereafter (**Supplemental Figure S1)**. As early as 24 h following peroral challenge, fecal Psae could be cultured from all mice with median loads of ∼105 CFU per g (**Figure 1A**). At later time points (48, 72, and 96 h, respectively), however, both fecal Psae positivity rates (88.9, 66.7, and 33.3%, respectively) as well as median Psae loads (p < 0.001; **Figure 1A**) were lower, whereas at the end of the experiment, Psae could be isolated from the ileal and colonic lumen in 22.2% and 33.3% of cases, respectively, (**Figure 1B**). Hence, only one third of mice with a human gut microbiota and subacute ileitis harbored MDR Psae in their intestinal tract 4 days post peroral challenge.

### Gut Microbiota Changes in Mice With a Human Gut Microbiota and Subacute Ileitis Following MDR *P. aeruginosa* Challenge

We next addressed whether MDR Psae challenge following subacute ileitis induction resulted in intestinal microbiota changes in mice with a human gut microbiota (**Figures 2A–H**). Quantitative culture-independent analysis revealed that until day 9 following ileitis induction mock treated hma mice displayed increased ileal loads of enterobacteria and enterococci (p < 0.05; **Figures 2B,C**), whereas both ileal Clostridium coccoides

FIGURE 2 | Ileal microbiota changes following MDR P. aeruginosa challenge of mice with a human gut microbiota suffering from subacute ileitis. Subacute ileitis was induced in mice harboring a human gut microbiota by peroral T. gondii infection on day (d) 0. At day 5 post ileitis induction mice were either perorally challenged with MDR P. aeruginosa (+Psae, dissolved in PBS) or with PBS alone (Mock). Uninfected mice with a human gut microbiota but without ileitis served as control animals (Naive). (A–H) The microbiota composition of the ileal lumen was determined 96 h later by quantitative Real-Time PCR amplifying bacterial 16S rRNA variable regions of the main intestinal bacterial groups (expressed as 16S rRNA gene numbers per ng DNA) including the total eubacterial load. Box plots represent the 75th and 25th percentiles of the median (black bar inside the boxes). Total range and significance levels determined by one-way ANOVA test followed by Tukey post-correction test for multiple comparisons are shown. Numbers of mice harboring respective bacterial groups in the ileal lumen out of the total numbers of analyzed animals are given in parentheses. Data were pooled from four independent experiments.

and Clostridium leptum group gene numbers were lower as compared to naive hma mice without ileitis (p < 0.001 and p < 0.005, respectively; **Figures 2G,H**). At day 9 post ileitis induction, lower Bacteroides / Prevotella species and Clostridium coccoides group loads could be detected in the ileal lumen of Psae challenged as compared to naive mice (p < 0.05; **Figures 2F,G**).

We additionally assessed the microbiota composition in the large intestinal tract (**Figures 3A–H**). Within 9 days following ileitis induction, total eubacterial loads increased in the colonic lumen of both mock and Psae challenged mice (p < 0.01 and p < 0.005, respectively; **Figure 3A**). This also held true for large intestinal enterobacteria (p < 0.005 and p < 0.001, respectively; **Figure 3B**) and enterococci (p < 0.01; **Figure 3C**), whereas Psae challenged animals displayed lower lactobacilli loads in their colon as compared to mock treated controls at day 9 p.i. (p < 0.05; **Figure 3D**). Hence, the intestinal loads of assessed bacterial taxa, groups and species did not differ between Psae challenged and unchallenged hma mice during subacute ileitis.

#### Macroscopic and Microscopic Inflammatory Sequelae of MDR *P. aeruginosa* Challenge in Mice With a Human Gut Microbiota Suffering From Subacute Ileitis

We next assessed whether peroral Psae challenge worsened subacute ileitis of hma mice. Mice of either treatment group displayed mean body weight loss of ∼5% during the observation period (not shown). At day 9 p.i., however, 58.8% of mock treated mice exhibited blood in their feces, whereas this was the case in 72.2% of Psae challenged and none of naive mice (**Figure 4A**). Following ileitis induction, mice of either cohort displayed comparable histopathological changes within the small intestinal mucosa and lamina propria (p < 0.005 vs. naive controls; **Figure 4B**). Of note, the range of histopathological scores was higher following Psae as compared to mock challenged mice at day 9 p.i., with single mice displaying even ileal necrosis in the former cohort, which was not the case in the latter group (**Figure 4B**).

FIGURE 3 | Colonic microbiota changes following MDR P. aeruginosa challenge of mice with a human gut microbiota suffering from subacute ileitis. Subacute ileitis was induced in mice harboring a human gut microbiota by peroral T. gondii infection on day (d) 0. At day 5 post ileitis induction mice were either perorally challenged with MDR P. aeruginosa (+Psae, dissolved in PBS) or with PBS alone (Mock). Uninfected mice with a human gut microbiota but without ileitis served as control animals (Naive). (A–H) The microbiota composition of the colonic lumen was determined 96 h later by quantitative Real-Time PCR amplifying bacterial 16S rRNA variable regions of the main intestinal bacterial groups (expressed as 16S rRNA gene numbers per ng DNA) including the total eubacterial load. Box plots represent the 75th and 25th percentiles of the median (black bar inside the boxes). Total range and significance levels determined by one-way ANOVA test followed by Tukey post-correction test for multiple comparisons are shown. Numbers of mice harboring respective bacterial groups in the colonic lumen out of the total numbers of analyzed animals are given in parentheses. Data were pooled from four independent experiments.

#### Intestinal Apoptotic, Proliferating, and Immune Cell Responses Upon MDR *P. aeruginosa* Challenge of Mice With a Human Gut Microbiota Suffering From Subacute Ileitis

We further expanded our histopathological analyses and quantitatively assessed apoptotic cells in the small intestinal epithelia applying in situ imunohistochemistry. Ileitis induction was accompanied by increased numbers of caspase3+ ileal epithelial cells (p < 0.001; **Figure 5A**; **Supplemental Figure S3A**). Of note, at day 9 p.i., Psae challenged mice were suffering from even more distinct ileal epithelial apoptosis as compared to mock treated mice (p < 0.01; **Figure 5A**; **Supplemental Figure S3A**).

We further quantitated Ki67+ cells in ileal epithelia indicative for proliferative/regenerative cell responses counteracting potential Psae-induced cell damage. Until day 9 p.i., Ki67+ cell numbers had markedly increased in the ileal epithelia as compared to naive controls (p < 0.001; **Figure 5B**; **Supplemental Figure S3B)**, but irrespective whether mice were Psae or mock challenged (n.s.; **Figure 5B**; **Supplemental Figure S3B**).

Given that T. gondii induced ileitis comprises a T celldriven immunopathological scenario (29), we next assessed T lymphocyte counts in ileal paraffin sections by in situ imunohistochemistry. At day 9 p.i., T. gondii infected mice displayed increased numbers of CD3+ cells in their ileal mucosa and lamina propria, but exhibited even higher T cell counts upon Psae as compared to mock challenge (p < 0.001; **Figure 5C**; **Supplemental Figure S3C**). Interestingly, increases in small intestinal T cell numbers following ileitis induction were paralleled by elevated numbers of FOXP3+ cells in the ileal mucosa and lamina propria of both, Psae and mock challenged mice at day 9 p.i. (p < 0.001; **Figure 5D**; **Supplemental Figure S3D)** with higher counts in the former as compared to the latter (p < 0.001; **Figure 5D**; **Supplemental Figure S3D)**.

Even though peroral T. gondii infection of susceptible mice is well-known to affect primarily the terminal ileum (29), we additionally included the large intestines into our immunohistopathological survey. Interestingly, ileitis induction was also accompanied by increases in apoptotic cell numbers in colonic epithelia (p < 0.001; **Figure 5E**; **Supplemental Figure S3E**) with a trend toward even higher counts in Psae as compared to mock treated mice at day 9 p.i. (n.s. due to high standard deviations). Like in the ileum, T. gondii infection resulted in increased Ki67+ colonic epithelial numbers (p < 0.001; **Figure 5F**; **Supplemental Figure S3F**), that were even more pronounced in Psae as compared to mock challenged mice (p < 0.01; **Figure 5F**; **Supplemental Figure S3F**). Furthermore, T. gondii induced elevated numbers of both CD3+ and FOXP3+ cells could be determined in the mucosa and lamina propria of the large intestines at day 9 p.i. (p < 0.001;

**Figures 5G,H**; **Supplemental Figures S3G,H**) with even higher counts in the Psae vs. mock cohort (p < 0.001; **Figures 5G,H**; **Supplemental Figures S3G,H**). Hence, Psae challenge resulted in more pronounced intestinal epithelial apoptotic and T cell responses upon ileitis induction that were not restricted to the ileum, but also involved the large intestines.

#### Intestinal Pro-Inflammatory Mediator Responses Upon MDR *P. aeruginosa* Challenge of Mice With a Human Gut Microbiota Suffering From Subacute Ileitis

We next addressed whether changes in apoptotic and immune cell responses upon Psae challenge of hma mice with subacute ileitis was accompanied by pro-inflammatory mediator responses in the intestinal tract. Irrespective whether Psae or mock challenged, hma mice displayed increased IFN-γ concentrations in ex vivo biopsies derived from the ileum, colon or MLN at day 9 p.i. (p < 0.001 vs. naive controls; **Supplemental Figures S4A–C**)**.** In addition, TNF and NO were multi-fold elevated in MLN following ileitis induction in either cohort (p < 0.005–0.001 vs. naive; **Supplemental Figures S4D,E)**. Hence, Psae challenge did not result in further augmented pro-inflammatory mediator responses in the intestinal tract following subacute ileitis induction of hma mice.

#### Extra-Intestinal Apoptotic, Proliferating, and Immune Cell Responses Upon MDR *P. aeruginosa* Challenge of Mice With a Human Gut Microbiota Suffering From Subacute Ileitis

We next addressed whether peroral MDR Psae challenge of hma mice with subacute ileitis might lead to more pronounced inflammatory responses in extra-intestinal compartments including liver, kidney, lung, and heart. Nine days following ileitis induction, hma mice exhibited increased numbers of apoptotic hepatic cells (p < 0.001 vs. naive; **Figure 6A**; **Supplemental Figure S5A)** that were even higher upon Psae as compared to mock challenge (p < 0.001; **Figure 6A**; **Supplemental Figure S5A)**. Increases in apoptotic cells were paralleled by elevated T lymphocyte and Treg counts in the livers of both, Psae and mock challenged mice at day 9

p.i. (p < 0.001; **Figures 6B,C**; **Supplemental Figures S5B,C** with higher numbers in the former as compared to the latter (p < 0.001 and p < 0.05, respectively; **Figures 6B,C**; **Supplemental Figures S5B,C**.

In the kidney, apoptotic cells, CD3+ and FOXP3+ cells increased upon ileitis induction of hma mice until day 9 p.i. (**Figures 6D–F**, **Supplemental Figures S5D–F**). Additional Psae challenge resulted in more pronounced increases in renal T lymphocytes as compared to the mock cohort (**Figure 6E**; **Supplemental Figure S5E**), whereas a trend toward higher Casp3+ and FOXP3+ cell counts could be observed in the kidneys following Psae challenge at day 9 p.i. (n.s.; **Figures 6D,F**, **Supplemental Figures S5D,F**).

Also in lung and heart, elevated apoptotic cell numbers could be observed 9 days following ileitis induction in either cohort (p < 0.005 and p < 0.05 vs. naive, respectively; **Figures 7A,D**, **Supplemental Figures S6A,D**) that were accompanied by multi-fold increased T lymphocyte counts in both organs (p < 0.001; **Figures 7B,E**, **Supplemental Figures S6B,E**). Remarkably, increases in T cell numbers in lung and heart were more pronounced in Psae as compared to mock challenged mice (p < 0.01 and p < 0.05, respectively; **Figures 7B,E**, **Supplemental Figures S6B,E**). Only in the lung, but not in the heart, higher Treg numbers could be assessed at day 9 p.i. (p < 0.001; **Figures 7C,F**, **Supplemental Figures S6C,F**) with similar counts in either cohort. Hence, additional peroral Psae challenge resulted in increased apoptotic and T cell responses in extra-intestinal compartments.

#### Extra-Intestinal Inflammatory Mediator Responses Upon MDR *P. aeruginosa* Challenge of Mice With a Human Gut Microbiota Suffering From Subacute Ileitis

We further assessed whether the observed increased apoptotic and immune cell responses in extra-intestinal compartments upon Psae challenge of hma mice with subacute ileitis were accompanied by pronounced inflammatory mediator secretion. Nine days following ileitis induction increased concentrations of pro-inflammatory cytokines such as IFN-γ and TNF could be observed in the liver of hma mice (p < 0.005–0.001; **Figures 8A,B**), but with no differences between mice of either cohort. Interestingly, hepatic concentrations of the antiinflammatory cytokine IL-10 were elevated in mock treated, but not Psae challenged mice at day 9 p.i. (p < 0.005 vs. naive, p < 0.05 vs. Psae; **Figure 8C**).

In the kidney, pro-inflammatory cytokines including IFN-γ, TNF, MCP-1, and IL-6 were increased to similar extent in Psae

and mock challenged mice at day 9 p.i. (p < 0.005–0.001 vs. naive; **Supplemental Figure S7**), which also held true for pulmonal IFN-γ, TNF, MCP-1, and IL-10 concentrations (p < 0.005–0.001 vs. naive; **Supplemental Figure S8**).

Hence, Psae challenge during subacute ileitis of hma mice did not result in further increased pro-inflammatory cytokine secretion in extra-intestinal organs, but led to a dampened antiinflammatory IL-10 response in the liver.

#### Systemic Pro-Inflammatory Mediator Responses Upon MDR *P. aeruginosa* Challenge of Mice With a Human Gut Microbiota Suffering From Subacute Ileitis

In the following we measured systemic cytokine secretion upon Psae challenge of hma mice during subacute ileitis. At day 9 p.i., increased pro-inflammatory mediators such as IFN-γ, TNF, and NO could be assessed in the spleen derived from mice of either cohort (p < 0.001; **Supplemental Figure S9**). In corresponding serum samples, increased IFN-γ, TNF, IL-6, MCP-1, and IL-10 concentrations were determined at day 9 p.i. (p < 0.05–0.001; **Figure 9**). Strikingly, even higher TNF and IL-6 levels could be measured in serum samples taken from Psae as compared to mock challenged hma mice with ileitis (p < 0.01; **Figures 9 B,C**). Of note, no viable bacteria that might have translocated from the intestinal tract to extra-intestinal and systemic tissue sites during subacute ileitis could be isolated from liver, kidney, lung, heart, spleen, or cardiac blood (not shown). Hence, Psae challenge of hma mice with induced subacute ileitis resulted in an augmented systemic pro-inflammatory cytokine response.

#### DISCUSSION

In recent years the global emergence of MDR Gram-negative species including Psae in humans as well as in livestock animals has come more and more into the focus of awareness (11, 30). In fact, the increasing prevalence of severe infections caused by MDR Gram-negative bacteria which allow for only very limited options to combat disease contributing to high fatality rates, has not only gained access to the conscience of medical professionals, but also to (health care) politicians as well as to the general public.

FIGURE 8 | Pro- and anti-inflammatory mediator responses in the liver following peroral MDR P. aeruginosa challenge of mice with a human gut microbiota suffering from subacute ileitis. At day 5 following subacute ileitis induction, mice with a human gut microbiota were either perorally challenged with MDR P. aeruginosa (+Psae, dissolved in PBS) or with PBS alone (Mock). Uninfected mice with a human gut microbiota but without ileitis served as control animals (Naive). 96 h following Psae challenge, (A) IFN-γ, (B) TNF, and (C) IL-10 concentrations were determined in hepatic ex vivo biopsies. Box plots represent the 75th and 25th percentiles of the median (black bar inside the boxes). The total range and significance levels determined by one-way ANOVA test followed by Tukey post-correction test for multiple comparisons are shown. The total number of analyzed animals are given in parentheses. Data were pooled from four independent experiments.

In consequence and of utmost importance, the WHO rated MDR Gram-negative species such as Psae as serious global threat to human health recently (4).

In healthcare settings, the risk of a patient for MDR Psae acquisition increases with the length of the hospital stay, most likely due to spread of the opportunistic pathogen from external sources such as contaminated surfaces and medical devices and/or via smear infection from body fluids or fecal samples derived from colonized/infected patients (5, 6, 31). Particularly information regarding prevalence of human intestinal Psae carriage are scarce, however, and range from 0 to 24% in healthy individuals (31–40), hence speaking against Psae as part of the commensal gut microbiota. One of the key factors facilitating stable intestinal (opportunistic) pathogenic colonization including Psae in mice and men, however, is antibiotic pre-treatment compromising the integrity of the complex gut microbiota, which subsequently provides open niches for the acquired bacteria and hence, potential internal sources for MDR Psae-associated infections lateron (8, 9, 41, 42). In fact, fecal Psae carriage before ICU admission has been shown to be associated with an ∼15-times increased risk for subsequent Psae-induced nosocomial infection (7), which might even more likely mount in a fatal outcome, given that particularly antibiotics-treated ICU patients with concomitant severe comorbidities including immunosuppression are prone to MDR Psae infection arising from the indigenous gut microbiota (3, 11, 43). It is therefore crucial to identify distinct risk factors including immunopathological conditions that might facilitate MDR Psae acquisition by the vertebrate host. Recent studies report higher abundances of Psae in the GIT of patients with underlying intestinal inflammatory diseases such as irritable bowel diseases or ulcerative colitis (40, 44). Interestingly, in our recent study MDR Psae could not only stably colonize the gut, but also induce overt inflammatory immune responses in intestinal, extra-intestinal and even systemic compartments upon peroral challenge of healthy WT mice that were lacking a gut microbiota due to preceding broad-spectrum antibiotic treatment (13). This was also the case, when (with respect to the gut microbiota) "humanized" and clinically uncompromised WT mice were subjected to the identical clinical MDR Psae strain (14).

In the present study, we had challenged hma mice with the opportunistic pathogen after subacute ileitis induction following peroral low-dose T. gondii infection—a non-lethal small intestinal inflammation model that has been established by us recently (19). Four days upon peroral application of a rather "supra-physiological" dose of 109 CFU, Psae could be isolated from the intestinal tract in approximately one third of hma mice with subacute ileitis and if so, in relatively low counts of <105 CFU per g luminal sample. In our previous study, intestinal MDR Psae could be cultured in up to 78% of healthy hma mice (i.e., without underlying intestinal inflammatory conditions) 4 weeks following peroral challenge with comparable loads (14). In line with results obtained from our mouse studies, fecal Psae counts were up to 5 log orders of magnitude lower in healthy human volunteers as compared to the bacterial counts within the oral suspension 6 days before (12).

Remarkably, irrespective of the (opportunistic) pathogenic bacterial carrier status, Psae-challenged mice appeared to display more pronounced clinical/macroscopic and microscopic features of subacute ileitis. Whereas, 72.2% of mice from the Psae cohort displayed macroscopic and/or microscopic abundance of blood in their feces 4 day after Psae challenge, this was only the case in 58.8% of mock counterparts. In support, the histopathological

test for multiple comparisons are shown. The total number of analyzed animals are given in parentheses. Data were pooled from four independent experiments.

changes in the ileal mucosa tended to be more severe in the Psae cohort as compared to the mock controls, given that individual mice exhibited even virtually fatal ileal mucosal changes such as transmural necrosis 4 days post Psae challenge, which could not be observed in the mock counterparts. The relatively high standard deviation in histopathological scores within the MDR Psae cohort points toward (yet unknown) distinct host factors that might determine the individual outcome of subacute ileitis upon Psae infection.

Furthermore, Psae induced a marked influx of T lymphocytes into the mucosa and lamina propria of both, the ileum and the colon that was accompanied by pronounced apoptotic responses in the ileal epithelia. Remarkably, the marked Psae-induced T cell responses were not restricted to the intestinal tract, but could also be observed in extra-intestinal compartments such as liver, kidney, lungs, and even the heart. In the liver, significant Psae-induced apoptotic changes could be further assessed and were accompanied by less distinct secretion of the anti-inflammatory cytokine IL-10 as compared to mock control mice. Strikingly, Psae challenged hma mice with subacute ileitis displayed even more pronounced systemic pro-inflammatory cytokine responses versus mock counterparts as indicated by higher TNF and IL-6 serum concentrations in the former as compared to the latter. In this context we were, however, not able to detect any viable bacteria originating from the gut microbiota including Psae that had translocated to extra-intestinal including systemic compartments. Nevertheless, it cannot be excluded that soluble bacterial factors including lipopolysaccharide derived from Gram-negative intestinal commensals or from MDR Psae might have found access to the circulation evoking systemic pro-inflammatory immune responses.

In support of our actual results, we demonstrated previously that peroral MDR Psae application to conventionally colonized IL-10−/− mice suffering from chronic colitis aggravated the outcome of the underlying intestinal immunopathological disease (16), again not only on the local (i.e., intestinal) level, but also beyond (i.e., in extra-intestinal and even systemic compartments).

Another recent study applying a lethal acute ileitis model induced by peroral high-dose (i.e., >50 cysts) T. gondii infection of hma mice revealed that the underlying acute gut inflammation facilitated Psae colonization (15). The severity of the gut inflammation model was, however, far too acute to decipher additional Psae induced inflammatory sequelae further accelerating the underlying (already hyper-acute) immunopathology (15).

Nevertheless, given the multitude of so far known Psae virulence factor it is not surprising that intestinal Psae carriage in health and disease mounts in immune responses accelerating the underlying disease. The Pseudomonas lipid A moiety, for instance, is part of the Gram-negative bacterial cell wall constituent lipopeptide and able to activate NFκB in a Toll-like receptor (TLR)−4 dependent fashion (45), which subsequently leads to recruitment of innate immune cells to the infection site, further perpetuating host immune responses in order to combat the (opportunistic) pathogenic challenge (1). In support, our previous studies revealed that, in fact, TLR-4 was involved in differentially mediating MDR Psae associated intestinal and extra-intestinal immune responses in IL-10−/− mice (46, 47).

The observed Psae induced immunopathological sequelae in hma mice with subacute ileitis -irrespective of the intestinal carrier status—are supported by our results derived from Campylobacter jejuni infection experiments indicating that a pathogen does not necessarily needs to be a permanent member of the intestinal ecosystem to induce pro-inflammatory responses (26, 48–50). More importantly, it is rather the initial hit set by the (opportunistic) pathogen that tips the balance toward immunopathology within the "ménage à trois" of (opportunistic) pathogens, commensal gut microbiota and host immunity (47, 48).

We further assessed whether Psae application resulted in changes of the gut microbiota composition applying cultureindependent methods, but could neither find any significant and biologically relevant Psae-induced changes in the luminal microbiota of the inflamed ileum, nor in the co-affected colon. One might, in fact, argue that the observation period of 4 days was far too short for overt changes in the intestinal ecosystem. Nevertheless, our actual results are supported by our previous study where even an intestinal carriage of MDR Psae by conventional IL-10−/− mice with chronic colitis for 6 weeks was not associated with significant changes in the gut microbiota composition as assessed by both, culture and molecular analyses (16).

In conclusion, our actual and previous studies revealed that peroral exposure to MDR Gram-negative opportunistic pathogenic species including Psae might worsen the outcome of underlying inflammatory conditions within the gut. In ongoing studies we are currently exploring antibiotics-independent factors preventing the susceptible host at risk including individuals suffering from inflammatory intestinal comorbidities from Psae-carriage and induced inflammation.

# AUTHOR CONTRIBUTIONS

MH: designed and performed experiments, analyzed data, wrote paper; UE and AG: performed experiments, analyzed data, co-edited paper; AK: analyzed data, co-edited paper; SB: provided advice in design and performance of experiments, co-edited paper.

#### FUNDING

This work was supported by grants from the German Research Foundation (DFG) to SB (SFB633, TP A7), MH (SFB633, TP B6 and SFB TR84, TP A5), and AK (SFB633, TP Z1), and from the German Federal Ministeries of Education and Research (BMBF) to SB and MH (PAC-Campy 01KI1725D).

#### ACKNOWLEDGMENTS

We thank Michaela Wattrodt, Ursula Rüschendorf, Alexandra Bittroff-Leben, Ines Puschendorf, Gernot Reifenberger, and the staff of the animal research facility of the Charité–University Medicine Berlin for excellent technical assistance, and animal breeding. We acknowledge support from the German Research Foundation (DFG) and the Open Access Publication Fund of Charité - Universitätsmedizin Berlin.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu. 2019.00049/full#supplementary-material

Supplemental Figure S1 | Experimental setup. The gut microbiota was depleted in conventional mice by broad-spectrum antibiotic treatment for 8 weeks. Three days before human fecal microbiota transplantation (FMT) the antibiotic cocktail was replaced by sterile tap water to assure antibiotic washout. Microbiota depleted animals were then subjected to human FMT on three consecutive days. In order to guarantee proper establishment of the complex human gut microbiota within the murine host, mice were kept for 10 days before subacute ileitis induction. At day 0, human microbiota associated (hma) mice were perorally subjected to low dose (i.e., one cyst) infection with T. gondii infection. 5 days later (d+5), hma mice with induced subacute ileitis were perorally challenged with a multi-drug resistant (MDR) P. aeruginosa (Psae) strain and followed up for another 4 days (d+9; necropsy).

Supplemental Figure S2 | Microbiota composition of human donor feces. Before human fecal microbiota transplantation of secondary abiotic mice on three consecutive days, main intestinal bacterial groups were quantitatively assessed in human fecal donor suspensions. 16S rRNA of the main intestinal bacterial commensals including enterobacteria (EB), enterococci (EC), lactobacilli (LB), bifidobacteria (Bif), Bacteroides / Prevotella species (B/P), Clostridium coccoides group (Clocc), Clostridium leptum group (Clept), and the total eubacterial load (TL) were analyzed by quantitative RT-PCR and expressed as gene numbers per ng DNA. Data shown are representative for four independent experiments. Total range as well as box plots representing the 75th and 25th percentiles of the median (black bar inside the boxes) are shown.

Supplemental Figure S3 | Representative photomicrographs depicting apoptotic and proliferating epithelial cell as well as immune cell responses in the ileum and colon following peroral MDR P. aeruginosa challenge of mice with a human gut microbiota suffering from subacute ileitis. At day 5 following subacute ileitis induction, mice with a human gut microbiota were either perorally challenged with MDR P. aeruginosa (ILE, Psae; dissolved in PBS) or with PBS alone (ILE, Mock). Uninfected mice with a human gut microbiota but without ileitis served as control animals (Naive). Representative photomicrographs out of four independent experiments illustrate the average numbers of apoptotic (Casp3+; A,E) and proliferating (Ki67+; B,F) epithelial cells as well as of T lymphocytes (CD3+; C,G) and regulatory T cells (FOXP3+; D,H) in at least six high power fields (HPF) that had been quantitatively assessed in ileal (A–D) and colonic (E–H) paraffin sections 96 h following Psae challenge applying in situ immunhistochemistry (100x magnification; scale bar: 100µm).

Supplemental Figure S4 | Intestinal pro-inflammatory mediator responses following peroral MDR P. aeruginosa challenge of mice with a human gut microbiota suffering from subacute ileitis. At day 5 following subacute ileitis

induction, mice with a human gut microbiota were either perorally challenged with MDR P. aeruginosa (+Psae, dissolved in PBS) or with PBS alone (Mock). Uninfected mice with a human gut microbiota but without ileitis served as control animals (Naive). 96 h following Psae challenge, IFN-γ concentrations were measured in ex vivo biopsies derived from the (A) ileum, (B) colon, and (C) mesenteric lymph nodes (MLN). In addition, (D) TNF and (E) nitric oxide levels were determined in MLN ex vivo biopsies. Box plots represent the 75th and 25th percentiles of the median (black bar inside the boxes). The total range and significance levels determined by one-way ANOVA test followed by Tukey post-correction test for multiple comparisons are shown. The total numbers of analyzed animals are given in parentheses. Data were pooled from four independent experiments.

Supplemental Figure S5 | Representative photomicrographs depicting apoptotic epithelial cell as well as immune cell responses in the liver and kidney following peroral MDR P. aeruginosa challenge of mice with a human gut microbiota suffering from subacute ileitis. At day 5 following subacute ileitis induction, mice with a human gut microbiota were either perorally challenged with MDR P. aeruginosa (ILE, Psae; dissolved in PBS) or with PBS alone (ILE, Mock). Uninfected mice with a human gut microbiota but without ileitis served as control animals (Naive). Representative photomicrographs out of four independent experiments illustrate the average numbers of apoptotic (Casp3+; A,D) epithelial cells as well as of T lymphocytes (CD3+; B,E) and regulatory T cells (FOXP3+; C,F) in at least six high power fields (HPF) that had been were quantitatively assessed in hepatic (A–C) and renal (D–F) paraffin sections 96 h following Psae challenge applying in situ immunhistochemistry (100x magnification; scale bar: 100µm).

Supplemental Figure S6 | Representative photomicrographs depicting apoptotic epithelial cell as well as immune cell responses in the lung and heart following peroral MDR P. aeruginosa challenge of mice with a human gut microbiota suffering from subacute ileitis. At day 5 following subacute ileitis induction, mice with a human gut microbiota were either perorally challenged with MDR P. aeruginosa (ILE, Psae; dissolved in PBS) or with PBS alone (ILE, Mock). Uninfected mice with a human gut microbiota but without ileitis served as control animals (Naive). Representative photomicrographs out of four independent experiments illustrate the average numbers of apoptotic (Casp3+; A,D) epithelial cells as well as of T lymphocytes (CD3+; B,E) and regulatory T cells (FOXP3+; C,F) in at least six high power fields (HPF) that had been were quantitatively assessed in pulmonal (A–C) and cardiac (D–F) paraffin sections 96 h following Psae challenge applying in situ immunhistochemistry (100x magnification; scale bar: 100µm).

Supplemental Figure S7 | Cytokine responses in the kidneys following peroral MDR P. aeruginosa challenge of mice with a human gut microbiota suffering from subacute ileitis. At day 5 following subacute ileitis induction, mice with a human gut microbiota were either perorally challenged with MDR P. aeruginosa (+Psae, dissolved in PBS) or with PBS alone (Mock). Uninfected mice with a human gut microbiota but without ileitis served as control animals (Naive). 96 h following Psae challenge, (A) IFN-γ, (B) TNF, (C) MCP-1, and (D) IL-6 concentrations were determined in renal ex vivo biopsies. Box plots represent the 75th and 25th percentiles of the median (black bar inside the boxes). The total range and significance levels determined by one-way ANOVA test followed by Tukey post-correction test for multiple comparisons are shown. The total numbers of analyzed animals are given in parentheses. Data were pooled from four independent experiments.

Supplemental Figure S8 | Cytokine responses in the lungs following peroral MDR P. aeruginosa challenge of mice with a human gut microbiota suffering from subacute ileitis. At day 5 following subacute ileitis induction, mice with a human gut microbiota were either perorally challenged with MDR P. aeruginosa (+Psae, dissolved in PBS) or with PBS alone (Mock). Uninfected mice with a human gut microbiota but without ileitis served as control animals (Naive). 96 h following Psae challenge, (A) IFN-γ, (B) TNF, (C) MCP-1, and (D) IL-10 concentrations were determined in pulmonal ex vivo biopsies. Box plots represent the 75th and 25th percentiles of the median (black bar inside the boxes). The total range and significance levels determined by one-way ANOVA test followed by Tukey post-correction test for multiple comparisons are shown. The total numbers of analyzed animals are given in parentheses. Data were pooled from four independent experiments.

Supplemental Figure S9 | Pro-inflammatory mediator responses in the spleen following peroral MDR P. aeruginosa challenge of mice with a human gut microbiota suffering from subacute ileitis. At day 5 following subacute ileitis induction, mice with a human gut microbiota were either perorally challenged with MDR P. aeruginosa (+Psae, dissolved in PBS) or with PBS alone (Mock). Uninfected mice with a human gut microbiota but without ileitis served as control animals (Naive). 96 h following Psae challenge, (A) IFN-γ, (B) TNF, and (C) nitric oxide concentrations were determined in splenic ex vivo biopsies. Box plots represent the 75th and 25th percentiles of the median (black bar inside the boxes). The total range and significance levels determined by one-way ANOVA test followed by Tukey post-correction test for multiple comparisons are shown. The total numbers of analyzed animals are given in parentheses. Data were pooled from four independent experiments.

### REFERENCES

of Campylobacter jejuni, microbiota and host innate immunity. PLoS ONE (2011) 6:e20953. doi: 10.1371/journal.pone.0020953

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Heimesaat, Escher, Grunau, Kühl and Bereswill. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Gut Microbiome as Target for Innovative Strategies Against Food Allergy

Roberto Berni Canani 1,2,3,4 \*, Lorella Paparo1,3, Rita Nocerino1,3, Carmen Di Scala1,3 , Giusy Della Gatta1,3, Ylenia Maddalena1 , Aniello Buono1,3, Cristina Bruno1,3, Luana Voto1 and Danilo Ercolini 4,5

*1 Department of Translational Medical Science – Pediatric Section, University of Naples "Federico II", Naples, Italy, 2 European Laboratory for the Investigation of Food-Induced Diseases, University of Naples "Federico II", Naples, Italy, 3 ImmunoNutritionLab at CEINGE-Advanced Biotechnologies, University of Naples "Federico II", Naples, Italy, 4 Task Force on Microbiome Studies, University of Naples "Federico II", Naples, Italy, 5 Department of Agricultural Sciences, University of Naples "Federico II", Naples, Italy*

#### Edited by:

*Nils Yngve Lycke, University of Gothenburg, Sweden*

#### Reviewed by:

*M. Cecilia Berin, Icahn School of Medicine at Mount Sinai, United States Javier Ochoa-Reparaz, Eastern Washington University, United States*

> \*Correspondence: *Roberto Berni Canani<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Mucosal Immunity, a section of the journal Frontiers in Immunology*

Received: *02 July 2018* Accepted: *22 January 2019* Published: *15 February 2019*

#### Citation:

*Berni Canani R, Paparo L, Nocerino R, Di Scala C, Della Gatta G, Maddalena Y, Buono A, Bruno C, Voto L and Ercolini D (2019) Gut Microbiome as Target for Innovative Strategies Against Food Allergy. Front. Immunol. 10:191. doi: 10.3389/fimmu.2019.00191* The dramatic increase in food allergy prevalence and severity globally requires effective strategies. Food allergy derives from a defect in immune tolerance mechanisms. Immune tolerance is modulated by gut microbiota function and structure, and microbiome alterations (dysbiosis) have a pivotal role in the development of food allergy. Environmental factors, including a low-fiber/high-fat diet, cesarean delivery, antiseptic agents, lack of breastfeeding, and drugs can induce gut microbiome dysbiosis, and have been associated with food allergy. New experimental tools and technologies have provided information regarding the role of metabolites generated from dietary nutrients and selected probiotic strains that could act on immune tolerance mechanisms. The mechanisms are multiple and still not completely defined. Increasing evidence has provided useful information on optimal bacterial species/strains, dosage, and timing for intervention. The increased knowledge of the crucial role played by nutrients and gut microbiota-derived metabolites is opening the way to a post-biotic approach in the stimulation of immune tolerance through epigenetic regulation. This review focused on the potential role of gut microbiome as the target for innovative strategies against food allergy.

Keywords: immune tolerance, gut microbiota, mediterranean diet, dysbiosis, probiotics, gut microbiota metabolites, short chain fatty acids, butyrate

#### INTRODUCTION

#### The Changing Scenario of Food Allergy

Food allergy (FA) is one of the most common allergic disorders in the pediatric age, and it has been considered as a global health problem, particularly in industrialized world (1). During the last two decades, studies have suggested that the epidemiology of FA has shown a dramatic increase in the prevalence, severity of clinical manifestations and risk of persistence into later ages, leading to an increase in medical visits, hospital admissions, treatments, burden of care on families, and economic impact, with an increase of costs for the families and healthcare system (2–4). According to the most recent epidemiological data, time trend analysis showed up to a 7-fold increase in hospital admissions for food severe allergic reactions in children in the UK, USA, Italy and Australia over the last 10 years (5–10). More than 170 foods have been identified as triggers of FA, such as tree nuts, eggs, peanuts, fish, shellfish, milk, wheat, soy, and seeds, with national and geographical variations concerning the most common FA (1, 10–15).

#### New Insights in the Pathogenesis of FA

FA derives from a breakdown of immune tolerance to dietary antigens (16). Immune tolerance mechanisms involved the activation of dietary antigens specific regulatory T cell (Tregs) (17). Current knowledge suggests that the epidemiology of FA may be influenced by epigenome-genome-environment interactions leading to an alteration of immune system function (18, 19). To stabilize or fall the prevalence of FA, new and innovative strategies to reduce FA incidence are required. Many factors have been postulated to contribute to the onset of FA. The multiple immutable risk factors that could influence FA onset include male sex, ethnicity (increased risk among Asian and African Americans children), and genetics (familial risk, human leukocyte antigen (HLA), and specific genes) (2, 20–25). In addition, there are other modifiable factors that can be potentially targeted to reduce or prevent FA. These factors are related (mode of delivery, breast milk, use of antibiotics or gastric acidity inhibitors, use of antiseptic agents, rural environment, junk food-based and/or low-fiber/high-fat diet, consumption of unpasteurized milk or fermented foods, exposure to pets), or unrelated (comorbid atopic dermatitis, timing and route of exposure to foods, reduced consumption of omega-3-polyunsaturated fatty acids or vitamin D insufficiency, antioxidants,) to an influence on gut microbiome development and function (26–40) (**Figure 1**).

#### Clinical Consequences of Gut Microbiome Dysbiosis in Children With FA

Many subjects with FA naturally outgrow it over time. Cow's milk allergy (CMA), hen's egg allergy and wheat allergy resolve in ∼50% of children by the age of 5–10 years. Other FAs (including peanuts, tree nuts, fish) have low rates of resolution and are considered persistent (41). In addition, many forms of FA, may be related with later development of other allergic manifestations such as oculorhinitis, atopic dermatitis, asthma, and urticaria (the so called "Atopic March") (42), as well as other diseases such as functional gastrointestinal disorders (FGIDs) (30, 43), inflammatory bowel diseases (IBD) (44), and psychiatric disorders, such as autistic spectrum disorders (ASD) attention deficit hyperactivity disorder (ADHD), and obsessivecompulsive disorder (OCD) (45). The pathogenesis of these events is still largely unknown, but increasing evidence suggest the hypothesis that a perturbation of gut microbiome, leading to alterations in immune system and gut-brain axis, could influence the occurrence of FA and FA-related conditions later in life (**Figure 2**).

#### Gut Microbiome Features in FA Investigating the Metagenomic and Metabolomics Features of Gut Microbiome

The knowledge and awareness of the roles played by gut microbiome and metabolites in the balance between health and disease is rapidly increasing. This is mainly due to advances in technology and the availability of high-sensitivity means to study microbial communities in any type of ecosystem. It is important for the clinicians and researchers dedicated to the FA field to know potential and limits of these technologies to better understand the value and significance of the findings reported in literature. **Box 1** summarizes terminology for gut microbiotabased investigations in FA.

Due to the power of genome DNA sequencing, we have learned much about the composition of gut microbial communities. In addition, the potential of transcriptomics, proteomics, and metabolomics are enlarging our understanding of the gut microbiota role in human health. Until the 1990s, knowledge of the gut microbiome was limited because the structure of gut microbiota was characterized using bacteriological culture. In the last decade, the composition of the gut microbiota was described by next generation sequencing of 16S ribosomal RNA genes. This is increasing the amount of information that can be retrieved by studying metagenomes from human samples, with the capability to infer the abundance of genes and potential metabolic pathways that characterize a microbial community. It is possible to describe the taxonomic composition of the microbiota and also to study the potential functions in a given system. Such methodological background is fundamental to investigate associations between microbiota structure and health as well as other environmental factors (46) and also to observe the changes of the gut microbiota in response to disease or perturbations in diet or lifestyle. An advanced technique to investigate gut microbiota at deep level is shotgun sequencing that represents a massive sequencing of the whole genome. Shotgun sequencing involves DNA random fragmentation, sequencing of these fragments and reconstruction of overlapping sequences to assemble them into a continuous sequence (47). Metabolomics represents one of the meta-omic approaches to study gut microbiota functions. Metabolomics uses high throughput techniques to characterize and quantify small molecules in several biofluids, such as feces, urine, plasma, serum, and saliva (48). The use of metabolomics is considered a powerful top-down systems biology approach, and it is essential to reveal the genetic-environment-health relationship, as well as the clinical biomarkers of diseases (49). Currently, the rapid development of several analytical platform, including liquid chromatography (LC), gas chromatography mass spectrometry (GC- MS), high-pressure LC (HPLC), ultra-pressure LC (UPLC), electrophoresis (CE) coupled to mass spectrometry (MS), Fourier transform infrared spectroscopy (FTIR), ion cyclotrone resonance-FT (ICR-FT), capillary and nuclear, and proton nuclear magnetic resonance spectroscopy (NMR-1H-NMR),

**Abbreviations:** FA, food allergy; CMA, cow's milk allergy; EHCF, extensively hydrolyzed casein formula; LGG, Lactobacillus rhamnosus GG; OIT, oral food immunotherapy; SU, sustained unresponsiveness; PBMCs, peripheral blood mononuclear cells; BLG, β lactoglobulin; OVA, ovalbumin; LAB, lactic acid bacteria; NDC, Non-digestible dietary carbohydrates; SCFAs, short chain fatty acids; Tregs, regulatory T cells; DCs, dendritic cells; Kyn, kynurenine; AhR, arylhydrocarbonreceptor; IPA, indole 3-propionic acid; I3A: indole-3-aldehyde; I3C, indole-3-carbinole.

allowed to better define bacteria related-metabolites and their metabolic pathways (50). **Box 2** summarizes techniques used to investigate the gut microbiota metagenomic and metabolomic features. Gut microbiota metabolomic features are still largely unexplored. Metabolomics will provide important insides in the pathogenesis of FA. In this light, preliminary data available on short chain fatty acids (SCFA) profile are opening new perspective of intervention (see below). What is needed is a transition from descriptive research to understanding the ways the microbiome interacts with the host and plays a role in health and disease. In this frame, controlled clinical interventions are of utmost importance to establish microbiota causative involvement and are the basis to implement approaches of personalized medicine (51, 52). The study of the relationship between microbiome and FA may begin with association and be translated to causation and clinical practice with appropriate advances in knowledge. Wide screening of microbial diversity in gut microbiome of patients with a sure diagnosis of FA, including a well-matched control population, may identify useful signatures in the microbiome that are specific for certain types of FA (53). If the wide screening included cohorts of patients with different dietary style or ethnicity, the common microbial signatures would be even stronger and provide a solid indication of the microbial biomarkers of FA. Further mapping of the genomic features associated with FA may be inferred by metagenomics and metabolomics, which may provide information on the functional microbial signatures associated with FA.

Biomarker strains or defined microbial systems may be tested in gnotobiotic or humanized animal models to observe the development of the disease, and beneficial vs. detrimental microbial metabolites can be recognized and used as final targets of microbiome-targeted personalized interventions. The identification of bacterial metabolites that positively affect the immune tolerance network, may be an interesting strategy against FA using a post-biotic approach.

#### Evidence on Gut Microbiome Dysbiosis in FA

Mounting evidence indicates that gut microbiome dysbiosis early in life represents a critical factor underlying FA (26, 27, 54, 55). Experimental data from animal models suggest a link between gut microbiome and the occurrence of FA. Tregs was found reduced in mice treated with antibiotic or

conditions, but recent evidence suggest the pivotal role of gut microbiome dysbiosis (induced by environmental factors). Emerging evidence support the hypothesis of dysbiosis as the first hit in the development of alterations in intestinal barrier and immune system function (responsible for the occurrence of FA and atopic march) and dysregulation of the brain-gut endocrine-immune system axis (responsible for the occurrence of FGIDs, IBD, and neuropsychiatric disorders), at least in part through an activation of epigenetic mechanisms.

#### Box 1 | A brief glossary for a better understanding of the potential of gut microbiota as target against food allergy.

in germ free mice, with consequent predisposition to allergy development (56–58). Administration of defined Clostridia, or bacteria-derived short-chain fatty acids (SCFA) to germ free mice induced an increase of Treg cells number, and reduced allergic response (56, 59–62). The allergy-protective action of Clostridia was also confirmed in the animal model, where a significant protective effect consisting in regulation of innate lymphoid cell function, Foxp3+ Tregs, immunoglobulin (Ig)A and intestinal epithelial permeability was demonstrated (63). A "humanized mice model," created with inoculation of microbiota-derived from human feces, resulted in an increase in Treg cells and a reduction of allergic symptoms (64). The functional role of dysbiosis associated with FA was also revealed by the different capacity of the gut microbiota of allergen-sensitized mice to increase Th2 cells number and IgE responses and to promote allergic sensitization (17).

Unfortunately, data characterizing the gut microbiome of patients affected by FA are still preliminary.

**Table 1** summarizes main evidence on FA-associated gut microbiome features. Heterogeneity in study design, used to define the gut microbiome, make it difficult to establish a causal relationship between development of FA and specific bacteria. Despite these limitations, at least four relevant observations on FA-associated gut microbiome can be raised:

Recent studies underline the importance of the modulation of gut microbiota through different dietary interventions in pediatric patients with FA. CMA children treated with soy and rice based

formula showed low fecal abundance of Coriobacteriaceae and Bifidobacteriaceae. Contrarily, Coriobacteriaceae, and certainly the genus Collinsella, the major bacteria that metabolized lactose in the gut, resulted increased in CMA children that consumed extensively hydrolyzed formula. In the same study, the authors found that fecal butyrate levels are positive correlated with abundance of Coriobacteriaceae (77). We showed that the treatment with extensively hydrolysed casein formula (EHCF) TABLE 1 | Main gut microbiome features in food allergy.

*FA, food allergy; FS, sensitization to food antigens; OTUs, operational taxonomic units; N.R., not reported; , increase; , decrease.*

containing the probiotic L. rhamnosus GG (LGG) in CMA children significantly increased SCFA-producers bacteria and butyrate fecal levels. These effects were associated with immune tolerance acquisition (76).

#### Targeting Gut Microbiome in FA The Importance of the Diet-Gut Microbiome Axis

Advances in metagenomics and metabolomics implicate diet and gut microbiome (the diet-gut microbiome axis) as key modulators of the maturation of the immune system. Findings from a recent systematic review further support the relationship between maternal diet during pregnancy and lactation and FA during childhood (78). Diet from conception (maternal diet) up to the first 24 months of age (baby diet), may influence the risk of developing FA (78–81). A recent study suggests that a healthy diet with high levels of fruits, vegetables and home-made foods is associated with less FA at the age of 24 months (82). Several studies have reported that nutrients impact the gut microbiota and the production of bacterial metabolites (83, 84). The Mediterranean diet (MD) is defined as a healthy balanced diet. It is characterized by high consumption of assorted cereals, legumes, fruits, vegetables, olive oil, and nuts; moderate consumption of red wine, poultry and fish, and a lower intake of red meat and sweets. MD during pregnancy and early life has been demonstrated to have a protective role against allergic disease in children (85). These effects could derive from the high intake of non-digestible dietary carbohydrates (NDC), the beneficial fatty acid profile that is rich in omega-3, the high levels of polyphenols, and other antioxidants (86). Nondigestible dietary carbohydrates represent the primary nutrient source for gut bacteria, and their fermentation leads to the production of SCFAs) (53, 87). It has been demonstrated that reduced availability of NDC lowered the concentration of fiberdegrading bacteria and increased mucin-degrading bacteria (88). High adherence to the MD has been associated with-increased levels of Prevotella bacteria and other Firmicutes and of SCFAs production (89). The immunomodulatory mechanisms elicited by SCFAs represent one of the strongest connections between diet, gut microbiome and allergic diseases (90). Major SCFAs included acetate, propionate, butyrate, and valerate (87). SCFAproducing bacteria represent a functional group, including Faecalibacterium prausnitzii and Eubacterium rectal, Roseburia are efficient butyrate producers (91). SCFAs are major energy source for colonocytes and influence epigenetically several non-immune (tight junction proteins, mucus production) and immune functions (macrophages, neutrophils, dendritic cells (DCs), T and B cells) involved in the immune tolerance network (92–98). SCFAs interaction with enterocytes are mediated by G-protein coupled receptors, namely GPCRs; GPR41, GPR43, GPR109A, and Olfr78) (99–101). GPR43 and GPR41 are highly expressed by enterocytes (102), whereas immune cells express GPR43 and GPR109A (100, 103–106). Among SCFAs, butyrate exerts a pivotal role in immune tolerance induction. It has been found that SCFAs are able to increase colonic Treg frequency and in vitro treatment of colonic Tregs, from germ free mice, with propionate significantly increased FoxP3 and IL-10 expression, a key cytokine that regulate Treg functions (60). Similarly, it has been demonstrated that butyrate facilitates generation of activated FoxP3+ Treg in mouse model (107).

Butyrate is able to regulate 103+DCs, reducing proinflammatory cytokines production and enhancing retinoic acid (RA) expression and subsequent generation of RA-regulated tolerogenic DCs (108). Butyrate promotes B cell differentiation and increases IgA and IgG production (107, 109).

The mechanisms are multiple and involve a strong epigenetic regulation of gene expression through the inhibition of histone deacetylase (HDAC) (60, 110, 111).

Butyrate deficiency has been observed in allergic children (112). Bacteria-produced SCFAs have been studies, has been specifically attributed to butyrate production by spore-forming Clostridiales. An enrichment of butyrate-producing taxa (Clostridia class and Firmicutes phylum) has been observed in children with faster CMA resolution (71). Altogether, these data suggest the potential of a "post-biotic" approach, based on the use of SCFAs against FA. In this light, data from our laboratory showed that oral butyrate induces a dramatic inhibition of acute allergic skin response, anaphylactic symptom score, body temperature decrease, intestinal permeability increase, anti-β lactoglobulin (BLG) IgE, IL-4, and IL-10 production in a murine model of CMA, suggesting a protective role of butyrate against FA (113).

We evaluated the direct effects of butyrate on peripheral blood mononuclear cells (PBMCs) from children affected by challengeproven IgE-mediated CMA. PBMCs were stimulated with BLG in the presence or absence of butyrate. Preliminary results showed that butyrate stimulates IL-10 and IFN-γ production and decreases DNA methylation rate of these two cytokine genes. The same effective butyrate dose induces FoxP3 demethylation and down-regulation of HDAC6/HDAC9 expression (113, 114). TABLE 2 | Main preclinical evidences on the probiotics role against food allergy.

Additional potential mechanisms by which diet could exert protolerogenic effects in the gut are related to the production of immunoregulatory metabolites, which interact with the host immune cells to promote non-responsiveness to innocuous luminal antigens (115). Tryptophan is an essential amino acid, which cannot be synthesized independently by humans; thus, it must be ingested through the diet. A portion of tryptophan is utilized to synthesize protein, and the other portion is catabolized to produce a variety of bioactive compounds, such as kynurenine (Kyn), serotonin and melatonin (84). Tryptophan absorbed by enterocytes directly activates the mTOR pathway by intracellular tryptophan receptors (116, 117). mTOR is known to play an important role in connecting metabolism and the immune system. During an inflammatory process, tryptophan is metabolized through the Kyn pathway. Kyn is an active metabolite and its biological activity is mediated by aryl hydrocarbon receptor (AhR) (118). The bond of Kyn to AhR receptor lead to the inhibition of DCs maturation and the proliferation of Th17 cells and Treg, increasing IL-22 and IL-10 production (119–122). Indole, indole 3 propionic acid (IPA) and indole-3-aldehyde (I3A) are produced by catabolism of tryptophan through intestinal commensal bacteria. A study demonstrated that strains of Clostridium cadaveris and Peptostreptococcus anaerobius CC14N metabolize tryptophan to produce IPA. Tryptophan can be also catabolized by lactobacilli to I3A. This metabolite protects gut mucosa against inflammation through AhR recognition (123). Indole-3-carbinole (I3C), an AhR ligand, has been demonstrated to boost immune tolerance in an ovalbumin (OVA)-sensitized mouse model (124). Mice fed I3C showed lower titres of anti-OVA IgG1 antibodies and higher number of CD103+MHC-II+ tolerogenic DCs compared to normal chow-fed control mice (124).

#### Engineering Gut Microbiome With Probiotics in FA

Immune tolerance is a major therapeutic target in FA. Evidence supports the concept that probiotics, defined as live microorganisms which when ingested in adequate amounts confer a beneficial effect on the host (125), could act at different levels in the immune tolerance network: modulating gut microbiota structure and function (increased production of butyrate) (53); interacting with enterocytes with subsequent modulation of non-immune (gut permeability and mucus thickness) (126–129) and immune tolerogenic mechanisms (stimulation of sIgA and β-defensins production) (130); modulation of cytokine response by immune cells (110–113, 131– 134). Main pre-clinical evidence on probiotic activity against

Box 3 | Targeting gut microbiota against FA: a research agenda.

immune system may occur directly through microbial components or indirectly through the action of metabolites, such as SCFAs. A positive modulation of this axis can counteract the pathogenesis of FA by promoting epithelial integrity, gut permeability, mucus production, CD103+ tolerogenic DCs, Treg differentiation, cytokines production, and sIgA release from plasma cells.

FA are summarized in **Table 2**. In the last decades, a number of experimental investigations have been developed to characterize organisms that could be used to modulate the immune system of patients with FA. Stimulation of human PBMCs with selected probiotic strains is a commonly used experimental tool for the investigation of the effect of these microorganisms on immune cells. The incubation of PBMCs with L. plantarum and B. adolescentis resulted in an increased production of the regulatory cytokine IL-10 by monocytes and DCs, and to enhanced IFN-γ production by T cells) (138, 148, 149). The addition of a probiotic mixture (L. casei W56, L. lactis W58, L. acidophilus W55, L. salivarius W57, B. infantis W52, B. lactis W18, and B. longum W51) to PBMCs from children with FA stimulated an increase of Th1 cells and related cytokines (141). An increase in T and B cells proliferation and a reduction in IgE production, were also observed in PBMCs from children with FA treated for 3 months with the same probiotic mixture (141). Using a 3D co-culture model of intestinal epithelial cells and PBMCs as an in vitro model of the intestinal mucosal immune system, Ghadimi et al. demonstrated that the probiotics B. breve and LGG inhibit activation of proinflammatory cytokines, IL-23, and IL-17, thereby reducing histone acetylation and simultaneously enhancing DNA methylation (135). The limitation of studying the effect of probiotics in vitro lies in the extrapolation of the results of in vivo benefits. For that reason, another commonly used experimental tool in this area is based on the use of animal model of FA. Using an OVA mouse model, it was demonstrated that oral administration of B. infantis reduced serum OVA-specific IgE, and IgG1 levels and Th2 cytokine release from splenocytes. Moreover, gut microbiota analysis showed that the probiotic-mediated protection was conferred by high abundance of Coprococcus and Rikenella (151). Different effects of oral administration of B. coagulans 09.712, L. plantarum 08.923, and B. infantis 11.322 in the reduction of Th2-driven intestinal inflammation and other symptoms associated with food-induced anaphylaxis, were demonstrated in a murine model of shrimp allergy (145). In particular, oral supplementation with B. coagulans 09.712 and L. plantarum 08.923 significantly ameliorates anaphylaxis symptoms and increases the population of CD4+ CD25+FoxP3+ T cells through mTORC inhibition, FoxP3 upregulation, and GATA-3 downregulation (145). Oral treatment with C. butyricum significantly ameliorated anaphylaxis symptoms and increased sIgA and FoxP3+Treg cells in the spleen from BLG-sensitized mice (150). Neonatal monocolonization of germ-free mice by L. casei BL2 modulated the allergic sensitization to cow's milk proteins, developed higher IgG responses against caseins, elicited by L. casei hydrolysed insoluble caseins into soluble immunogenic peptides (152). Similar results were obtained by others who observed a decrease of concentrations of IgE, IL-4, and IL-13 following administration of B. infantis CGMCC313-2 in BLG-sensitized mice (153). Oral administration of VSL#3 (a mixture of Streptococcus thermophilus BT01, B. breve BB02, B. longum BL03, B. infantis BI04, L. acidophilus BA05, L. plantarum BP06, L. paracasei BP07, L. delbrueckii subsp. bulgaricus BD08) to sensitized mice significantly reduces Th2 immune responses and protects against anaphylactic reactions in a mouse model of FA (154). Also, the incubation of mouse spleen cells from sensitized mice with probiotic mixture reduced allergen- stimulated IL-13 and IL-5 production and increased of IFN-γ and IL-10 production (154). An immunoregulatory action by LGG has been demonstrated in a murine model of CMA. LGG administration suppressed Th2 responses, such as reduced hypersensitivity score and lowered serum CMPspecific IgG1, while promoting IFN-γ and CMP-specific IgG2a levels (155). Similar results have been reported by our group in a BLG-sensitized mice model, in which we found that the administration of LGG added to EHCF elicited a significant

reduction of allergic reaction, and of IL-4, IL-5, IL-13 and specific IgE production (139).

Clinical studies have investigated the efficacy of selected probiotic strains against FA. The effect appears strain-specific and more evident in the pediatric age group. In a randomized double-blind placebo-controlled trial, it was demonstrated that the administration of L. casei CRL431 and B. lactis BB12 added to hypoallergenic formula for 12 months did not modulate the rate of immune tolerance acquisition to cow's milk proteins in infants with CMA (140). Using a similar study design, we have demonstrated that EHCF containing the probiotic LGG is able to accelerate immune tolerance acquisition in CMA children. Children (aged 1–12 months), consecutively referred for suspected CMA (IgE- or non-IgE-mediated), but still receiving cow's milk proteins, were evaluated in the study. Subjects were randomly allocated to one of the two groups of dietary interventions: EHCF (control group); and EHCF containing LGG (at least 1.4×107 CFU/100 mL; active group). After 12 months, the double-blind placebo- controlled food challenge was negative in 15 of 28 control infants (53.6%) and in 22 of 27 infants receiving EHCF with LGG [(81.5%, p = 0.027)] (156). The results were confirmed in a subsequent trial, when the effect of 5 different dietary strategies was investigated: EHCF, EHCF + LGG, partially hydrolyzed rice formula, soy formula or amino acid-based formula, in children affected by IgE- or non-IgE-mediated CMA. After the treatment period of 12 months, the proportion of children acquiring immune tolerance to cow's milk proteins was significantly higher in the group receiving EHCF+LGG (78.9%) than in other groups (157). At the 3-year follow- up of another pediatric cohort, a further confirmation of a greater rate of resolution of IgE-mediated CMA as well as a lower incidence of other atopic manifestations was described after treatment with EHCF+LGG (158). These effects could derive at least in part by a modulation elicited by selected LGG components on immune functions through different pathways including enterocytes, monocytes, mast cells, DCs and Tregs (159–162), and by an expansion of butyrate- producing gut microbiota (53, 76). Accordingly, studies in children with eczema and/or CMA who received EHCF plus LGG showed benefits in decreasing inflammation and gastrointestinal symptoms (163). Probiotics have been also proposed to reinforce the effectiveness of immunotherapy (164). Oral food immunotherapy (OIT) is

#### REFERENCES

currently the most investigated approach for persistent FA and it is based on the concept that repeated oral/intestinal exposures to antigens normally leads to tolerance. Randomized doubleblind placebo- controlled trial was performed in 62 children with peanut allergy treated with fixed dose of probiotic together with peanut OIT (PPOIT) or placebo once daily for 18 months (165). Sustained unresponsiveness (SU), determined by double blind placebo controlled food challenge (DBPCFC), was achieved in 82.1% of children receiving PPOIT compared with 3.6% of those receiving placebo. PPOIT also induced high rates of resolution (90%) and was associated with reduced skin prick test reactivity, decreased peanut-specific IgE and increased peanutspecific IgG4 levels. No participants withdrawing because of adverse reactions.

No OIT control group was evaluated to determine if the probiotic itself had any effect on SU (165). Further studies are required to evaluate this approach comparing peanut OIT and probiotics with peanut OIT with placebo or probiotic alone.

### CONCLUSIONS

Gut microbiome could be a promising target for innovative therapeutic and preventive strategies against FA. The results of the studies are encouraging, but more data are needed to better define the potential of modulating the diet-gut microbiome–immune system axis to fight FA (**Figure 3**). We are approaching a new era in which we can regulate immune system development and function through dietary intervention and measure the clinical impact through gut microbes and their metabolites. Given the current gaps in the investigational approaches and data analysis and interpretation, we need more scientific evidence that can be translated in clinical practice (**Box 3**).

Understanding how nutrients and metabolites, or probiotics could influence gut bacteria communities and the immune system will contribute to building up a precision medicine approach for FA care (**Figure 4**).

# AUTHOR CONTRIBUTIONS

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

165. Tang ML, Ponsonby AL, Orsini F, Tey D, Robinson M, Su EL, et al. Administration of a probiotic with peanut oral immunotherapy: a randomized trial. J Allergy Clin Immunol. (2015) 135:737–44. doi: 10.1016/j.jaci.2014.11.034

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Berni Canani, Paparo, Nocerino, Di Scala, Della Gatta, Maddalena, Buono, Bruno, Voto and Ercolini. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Pituitary Adenylate Cyclase-Activating Polypeptide—A Neuropeptide as Novel Treatment Option for Subacute Ileitis in Mice Harboring a Human Gut Microbiota

Stefan Bereswill 1 , Ulrike Escher 1 , Anne Grunau1 , Anja A. Kühl 2 , Ildiko R. Dunay 3 , Andrea Tamas 4 , Dora Reglodi 4 and Markus M. Heimesaat 1 \*

1 Department of Microbiology, Infectious Diseases, and Immunology, Charité—Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany, 2 Department of Medicine I for Gastroenterology, Infectious Diseases and Rheumatology/Research Center ImmunoSciences (RCIS), Charité—Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany, 3 Medical Faculty, Institute of Inflammation and Neurodegeneration, University Hospital Magdeburg, Magdeburg, Germany, 4 Department of Anatomy, MTA-PTE PACAP Research Team, Centre for Neuroscience, University of Pecs Medical School, Pecs, Hungary

#### Edited by:

Ashutosh K. Mangalam, The University of Iowa, United States

#### Reviewed by:

Gianluca Matteoli, KU Leuven, Belgium Baskar Balakrishnan, Mayo Clinic, United States

\*Correspondence: Markus M. Heimesaat<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Mucosal Immunity, a section of the journal Frontiers in Immunology

Received: 30 May 2018 Accepted: 01 March 2019 Published: 22 March 2019

#### Citation:

Bereswill S, Escher U, Grunau A, Kühl AA, Dunay IR, Tamas A, Reglodi D and Heimesaat MM (2019) Pituitary Adenylate Cyclase-Activating Polypeptide—A Neuropeptide as Novel Treatment Option for Subacute Ileitis in Mice Harboring a Human Gut Microbiota. Front. Immunol. 10:554. doi: 10.3389/fimmu.2019.00554 The neuropeptide Pituitary adenylate cyclase-activating polypeptide (PACAP) is well-known for its important functions in immunity and inflammation. Data regarding anti-inflammatory properties of PACAP in the intestinal tract are limited, however. In our present preclinical intervention study we addressed whether PACAP treatment could alleviate experimental subacute ileitis mimicking human gut microbiota conditions. Therefore, secondary abioitic mice were subjected to human fecal microbiota transplantation (FMT) and perorally infected with low-dose Toxoplasma gondii to induce subacute ileitis on day 0. From day 3 until day 8 post-infection, mice were either treated with synthetic PACAP38 or placebo. At day 9 post-infection, placebo, but not PACAP treated mice exhibited overt macroscopic sequelae of intestinal immunopathology. PACAP treatment further resulted in less distinct apoptotic responses in ileal and colonic epithelia that were accompanied by lower T cell numbers in the mucosa and lamina propria and less secretion of pro-inflammatory cytokines in intestinal ex vivo biopsies. Notably, ileitis-associated gut microbiota shifts were less distinct in PACAP as compared to placebo treated mice. Inflammation-ameliorating effects of PACAP were not restricted to the intestines, but could also be observed in extra-intestinal including systemic compartments as indicated by lower apoptotic cell counts and less pro-inflammatory cytokine secretion in liver and lungs taken from PACAP treated as compared to placebo control mice, which also held true for markedly lower serum TNF and IL-6 concentrations in the former as compared to the latter. Our preclinical intervention study provides strong evidence that synthetic PACAP alleviates subacute ileitis and extra-intestinal including systemic sequelae of T cell-driven immunopathology. These findings further support PACAP as a novel treatment option for intestinal inflammation including inflammatory bowel diseases (IBD).

Keywords: pituitary adenylate cyclase-activating polypeptide (PACAP), subacute ileitis, Th1-type immunopathology, human fecal microbiota transplantation, gut-brain axis, preclinical intervention study

# INTRODUCTION

The Pituitary adenylate cyclase-activating polypeptide (PACAP) could be first identified in the hypothalamus exerting adenylate cyclase stimulating activity within the pituitary gland (1). Being part of the vasoactive intestinal peptide (VIP)/secretin/glucagon family, the neuropeptide shares 68% sequence homology with VIP and presents with two biologically active amidated forms (i.e., PACAP 27 and PACAP38) after alternative splicing from its pre-pro precursor (1, 2). Beyond the nervous system, PACAP expression can be found in many peripheral organs within the reproductive, respiratory, endocrine and digestive system as well as in lymphoid compartments including immune cells (2). PACAP is able to bind to VPAC1, VPAC2, and PAC1 receptors on innate immune cells including macrophages and lymphocytes (3–5). Given its virtual ubiquitous expression, PACAP presents with a variety of cyto-protective properties including anti-inflammatory and anti-apoptotic effects (5, 6). In experimental models of encephalomyelitis and arthritis, for instance, distinct anti-inflammatory effects following exogenous PACAP application have been demonstrated (7, 8). Data regarding inflammation-ameliorating properties of synthetic PACAP in the gastrointestinal tract are limited, however. PACAP−/− mice suffered from more severe acute colitis following dextran sodium sulfate (DSS) challenge as compared to wildtype counterparts (9, 10).

Human inflammatory bowel diseases (IBD) such as Crohn's disease and ulcerative colitis comprise chronic inflammatory conditions with acute episodes within the gastrointestinal tract and are of multi-factorial etiology (11–13). Most in vivo studies mimicking human IBD have applied experimental models of the large intestines so far, whereas, however, small intestinal inflammation models are rather scarce (14).

In our previous studies we applied an acute ileitis model characterized by a severe T cell-driven immunopathology with a lethal outcome within 1 week after peroral high-dose (i.e., >50 cysts) Toxoplasma gondii infection of mice (14–17). This highdose T. gondii infection model mimics key features of the acute phase of human Crohn's disease ("ileitis terminalis"), given (i) the predilection site of the terminal ileum, (ii) the underlying T helper cell (Th)−1 immunopathology that is (iii) associated with marked shifts in gut microbiota composition (dysbiosis) toward Gram-negative gut commensals, (iv) further contributing to an acceleration of the inflammatory scenario via Toll-like receptor (TLR)−4 dependent signaling of lipopolysaccharide (LPS) derived from the Gram-negative commensal bacterial cell walls (14). We were able to show that treatment of mice with synthetic PACAP could efficiently ameliorate acute ileitis and even extra-intestinal sequelae of T. gondii infection in a timeof-treatment dependent fashion with highest efficacy during a prophylactic regimen when starting PACAP application prior ileitis induction (17). The within 1 week lethal outcome of the hyper-acute inflammatory scenario following high-dose T. gondii infection needs to be considered as a limitation of the applied gut inflammation model, however.

This prompted us to unravel potential immune-modulatory properties of PACAP during small intestinal inflammation of less acute severity. Since the host specific gut microbiota is known to be essentially involved in the onset, progress, and outcome of distinct immunopathological conditions including intestinal inflammation (15, 18, 19), we generated (with respect to their gut microbiota) "humanized" mice. Very recently we were able to show that within 9 days following peroral infection with a low-dose (i.e., 1 cyst) of T. gondii, mice harboring a human gut microbiota develop non-lethal subacute ileitis characterized by increased T cell-dependent gut epithelial apoptosis and pro-inflammatory cytokine secretion in intestinal and extra-intestinal compartments (20). Furthermore, low-dose T. gondii infected mice displayed rather mild-to-moderate histopathological changes of the ileal mucosa and lamina propria, whereas no transmural small intestinal necrosis like in the lethal high-dose infection model could be observed. In the present preclinical intervention study we assessed whether therapeutic PACAP application starting 3 days after ileitis induction (i) resulted in disease-alleviating effects in the intestinal tract, (ii) was associated with distinct shifts in gut microbiota composition, and furthermore, (iii) whether potential PACAP-induced antiinflammatory effect could also be observed in extra-intestinal organs or (iv) even in systemic compartments.

### MATERIALS AND METHODS

#### Generation of Mice With a Human Gut Microbiota by Fecal Microbiota Transplantation

Female C57BL/6j mice were raised and maintained under specific pathogen-free (SPF) conditions in the Forschungseinrichtungen für Experimentelle Medizin (FEM, Charité - University Medicine, Berlin, Germany). Mice with a depleted microbiota (i.e., secondary abiotic mice) were generated as reported earlier (15, 18). Briefly, eight-week-old mice were kept in autoclaved cages and treated with an antibiotic cocktail for 8 weeks containing ampicillin plus sulbactam (1 g/L; Ratiopharm, Germany), vancomycin (500 mg/L; Cell Pharm, Germany), ciprofloxacin (200 mg/L; Bayer Vital, Germany), imipenem (250 mg/L; MSD, Germany) and metronidazole (1 g/L; Fresenius, Germany) (ad libitum). Successful depletion of the gut microbiota was confirmed in fecal samples by both, culture and molecular (16S rRNA based) methods as stated elsewhere (18, 21). In order to guarantee antibiotic washout, the antibiotic cocktail was replaced by sterile tap water (ad libitum) 3 days before human fecal microbiota transplantation (FMT). Fresh fecal samples that were negative for enteropathogenic bacteria, viruses, and parasites were donated from five healthy human individuals, dissolved in sterile phosphate buffered saline (PBS; Gibco, Life Technologies, United Kingdom) and stored

**Abbreviations:** CFU, colony forming units; DSS, dextran sodium sulfate; FMT, fecal microbiota transplantation; H&E, hematoxylin and eosin; Hma, human microbiota associated; HPF, high power field; IBD, inflammatory bowel disease; IFN, interferon; IL, interleukin; i.p., intraperitoneal; MLN, mesenteric lymph nodes; PACAP, Pituitary adenylate cyclase-activating polypeptide; PBS, phosphate buffered saline; p.i., post-infection; PLC, placebo; qRT-PCR, quantitative real-time polymerase chain reaction; SPF, specific pathogen free; Th, T helper cell; TNF, tumor necrosis factor; VIP, vasoactive intestinal peptide.

at −80◦C until usage as described previously (18). Immediately before FMT, individual fecal aliquots were thawed, pooled and applied to secondary abiotic mice by gavage on three consecutive days (18). Between individual FMT challenges, bacterial loads varied <0.5 orders of magnitude (**Figure S1**). For appropriate establishment of the complex human gut microbiota within the murine host, mice were kept for 12 days before subacute ileitis induction. Immediately before peroral T. gondii infection (d0) and at the end of the observation period [i.e., day 9 post-infection (p.i.)] individual fecal samples were collected from human microbiota associated (hma) mice for subsequent quantification of the main gut bacterial groups by molecular methods as described elsewhere (15, 18, 19).

### Induction of Subacute Ileitis, Determination of Parasitic Loads

For induction of subacute ileitis, hma mice were perorally challenged with one cyst of T. gondii strain ME49 in 0.3 mL brain suspension by gavage as reported earlier (20). T gondii DNA was quantitated in ileal ex vivo biopsies as stated previously (16).

#### Treatment

PACAP38 was synthesized at the Department of Medical Chemistry, University of Szeged (Hungary) and applied to mice with a daily dose of 1.5 mg per kg body weight (in PBS) (9, 17). Mice received either the synthetic PACAP38 or PBS as placebo control (PLC) via the intraperitoneal (i.p.) route (0.3 mL) from day 3 p.i. until day 8 p.i. once daily.

# Clinical Conditions and Sampling Procedures

Clinical conditions of mice including body weight loss were monitored daily. Nine days post ileitis induction mice were sacrificed by isoflurane treatment (Abbott, Germany). Cardiac blood and ex vivo biopsies were derived from mesenteric lymph nodes (MLN), lung, liver, ileum and colon under aseptic conditions. Respective tissue samples were taken from each mouse in parallel for immunological, immunohistochemical, and microbiological analyses. Whereas the small intestinal lengths were assessed by measuring the distance from the duodenum leaving the stomach to the ileal-cecal valve, the colonic lengths were measured from the ileal-cecal valve to the rectum with a ruler and expressed in cm.

# Histopathology and Immunohistochemistry

Ex vivo biopsies were derived from the terminal ileum, colon, lung, and liver, fixed in 5% formalin and embedded in paraffin. Histopathological changes were quantitated in 5µm thin, with hematoxylin and eosin (H&E) stained ileal paraffin sections applying a standardized histopathological scoring system ranging from 0 to 6 as stated elsewhere (15).

Paraffin sections (5µm) were further analyzed applying in situ immunohistochemistry as reported previously (22). Briefly, in order to quantitate apoptotic cells and T lymphocytes, primary antibodies against cleaved caspase-3 (Asp175, #9661, Cell Signaling, Leiden, Netherlands; 1:200), and CD3 (#IR50361- 2, Dako, Santa Clara, CA, USA; 1:5) were applied, respectively. The average number of positively stained cells (within at least six high power fields (HPF), 0.287 mm2 , 400x magnification) was assessed by an independent and blinded investigator.

# Pro-inflammatory Cytokine Secretion

Ileal and colonic tissue samples (∼1 cm2 ) were cut longitudinally and washed in PBS. Respective intestinal ex vivo biopsies as well as samples derived from MLN (3 lymph nodes), liver samples (∼1 cm2 ) and lung were placed in 24-flat-bottom wellculture plates (Falcon, Germany) supplemented with 500µL serum-free RPMI 1640 medium (Gibco, life technologies), penicillin (100 U/mL, Biochrom, Germany) and streptomycin (100µg/mL; Biochrom). After overnight incubation at 37◦C, culture supernatants were taken and tested for IL-6, TNF, and IFN-γ secretion applying the Mouse Inflammation Cytometric Bead Assay (CBA; BD Bioscience) on a BD FACSCanto II flow cytometer (BD Bioscience). Systemic pro-inflammatory cytokine concentrations were measured in serum samples.

#### Molecular Analysis of the Human Fecal Donor Suspensions and the Intestinal Microbiota

Human fecal donor suspensions as well as fresh ileal and colonic luminal samples were immediately transferred to liquid nitrogen and stored at −80◦C until further analyses. Fecal DNA extraction was performed as reported earlier (15). Briefly, the amount of DNA was assessed with a Quant-iT PicoGreen reagent (Invitrogen, UK) and adjusted to 1 ng per µL. The main human gut bacterial groups including enterobacteria, enterococci, lactobacilli, bifidobacteria, Bacteroides/Prevotella species, Clostridium coccoides group, and Clostridium leptum group as well as the total eubacterial loads were determined applying quantitative real-time polymerase chain reaction (qRT-PCR) and species-, genera- or group-specific 16S rRNA gene primers (Tib MolBiol, Germany) as indicated (**Figure S2**) and further described previously (19, 23) (expressed as 16S rRNA gene copies per ng DNA).

# Bacterial Translocation

In order to survey viable bacteria translocating from the gastrointestinal tract to extra-intestinal including systemic tissue sites, ex vivo biopsies were taken from MLN, lungs, and liver, homogenized in sterile PBS and analyzed in serial dilutions on defined solid culture media as reported previously (15, 24). Cardiac blood was incubated in thioglycolate enrichment broths (BD Bioscience, Germany) for at least 7 days at 37◦C and then streaked on culture media (25, 26). For at least 2 days bacteria were grown under aerobic, microaerobic and anaerobic conditions at 37◦C.

# Statistical Analysis

Medians and levels of significance were determined by the one-way ANOVA test followed by Tukey post-correction test for multiple comparisons. Two-sided probability (p) values ≤0.05 were considered significant. Experiments were reproduced three times.

FIGURE 1 | Macroscopic effects in PACAP treated mice with a human gut microbiota suffering from subacute ileitis. Subacute ileitis was induced by T. gondii infection of mice harboring a human gut microbiota (day 0). Starting 3 days post-infection (p.i.), mice were either treated with PACAP or placebo (PLC). Uninfected mice with a human microbiota served as control animals (Naive). At day 9 p.i., (A) relative body weight loss, (B) small intestinal, and (C) large intestinal lengths were assessed. Box plots represent the 75 and 25th percentiles of the median (black bar inside the boxes). The total range and significance levels determined by one-way ANOVA test followed by Tukey post-correction test for multiple comparisons are shown. Total numbers of analyzed animals are given in parentheses. Data were pooled from four independent experiments.

#### RESULTS

#### Macroscopic Sequelae in PACAP Treated Mice With a Human Gut Microbiota Suffering From Subacute Ileitis

In the present preclinical intervention study we addressed whether therapeutic application of synthetic PACAP dampened pro-inflammatory responses in intestinal and extra-intestinal including systemic compartments of mice with a human gut microbiota suffering from subacute ileitis. The small intestinal immunopathology was induced by peroral low dose T. gondii infection on day 0 (20). Three days post ileitis induction, hma mice were treated with synthetic PACAP for 6 days in total and clinical conditions including the body weights were monitored. Remarkably, T. gondii infected placebo (PLC) control mice exhibited substantial body weight loss until day 9 p.i., whereas this was not the case in PACAP treated mice (**Figure 1A**). Given that intestinal inflammation is accompanied by a significant shortening of the inflamed intestine (15, 18, 27), we measured small intestinal lengths upon necropsy. At day 9 p.i., PACAP treated mice displayed slightly longer small intestines as compared to PLC control animals (**Figure 1B**). Of note, the standard deviation within the PLC group was relatively high, which might explain the non-significant differences compared to the naive cohort (**Figure 1B**). Even though the terminal ileum is well-known to be the predilection site of T. gondii induced immunopathology (14), we also assessed the lengths of the large intestines. Interestingly, PLC, but not PACAP treated mice displayed significantly shorter large intestines at day 9 p.i. as compared to naive mice (**Figure 1C**), pointing toward inflammatory responses beyond the terminal small intestine, additionally affecting the large intestinal tract. Hence, PACAP treatment resulted in a better clinical / macroscopic outcome of T. gondii induced subacute ileitis. To rule out that the observed differences in disease outcomes might be due to different T. gondii infection efficiencies, we assessed parasitic DNA concentrations in ileal ex vivo biopsies at day 9 p.i., but detected comparable ileal T. gondii loads in mice of the PACAP and PLC cohort (not shown).

#### Intestinal Anti-inflammatory Effects in PACAP Treated Mice With a Human Gut Microbiota Suffering From Subacute Ileitis

We next addressed PACAP-induced anti-inflammatory effects in the intestinal tract on microscopic level. At day 9 following ileitis induction ileal histopathological scores were lower in PACAP treated hma mice as compared to PLC control animals (**Figure 2**). Of note, we could not observe significant differences in histopathological changes in the mucosa and lamina propria of H&E stained colonic paraffin sections (not shown). Since apoptosis is regarded a reliable parameter for the histopathological grading of intestinal inflammation (16), we further assessed T. gondii induced apoptotic cell responses in the intestines by quantification of caspase3+ intestinal epithelial cells applying in situ immunohistochemistry. At day 9 p.i., hma mice displayed multifold increased numbers of apoptotic ileal epithelial cells (**Figure 3A**). These increases were, however, less pronounced in PACAP treated mice (**Figure 3A**; **Figure S3A**).

Given that T lymphocytes are the major driving forces of T. gondii induced ileitis (14), we additionally stained ileal paraffin sections with CD3 antibodies. Ileitis induction was, in fact, accompanied by a marked increase in CD3+ ileal epithelial cell numbers until day 9 p.i. (**Figure 3B**, **Figure S3B**), but to a

FIGURE 2 | Microscopic effects in PACAP treated mice with a human gut microbiota suffering from subacute ileitis. Subacute ileitis was induced by T. gondii infection of mice harboring a human gut microbiota (day 0). Starting 3 days post-infection (p.i.), mice were either treated with PACAP or placebo (PLC). Uninfected mice with a human microbiota served as control animals (Naive). At day 9 p.i., histopathological changes within the ileum were quantitated in H&E stained ileal paraffin sections applying a standardized histopathological scoring system (left). Box plots represent the 75 and 25th percentiles of the median (black bar inside the boxes). The total range and significance levels determined by one-way ANOVA test followed by Tukey post-correction test for multiple comparisons are shown. Total numbers of analyzed animals are given in parentheses. Data were pooled from four independent experiments. Histopathological changes are illustrated in photomicrographs representative for four independent experiments (right; 100x magnification, scale bar 100µm).

significantly lesser extent upon PACAP treatment (**Figure 3B**, **Figure S3B**). Remarkably, T. gondii-induced increases in both, caspase3+ and CD3+ cells could also be observed in the epithelia and mucosa / lamina propria of the large intestines, respectively (**Figures 3C,D, Figures S3C,D**). Like in the ileal compartment, PACAP treatment was accompanied with significantly less distinct apoptosis and abundance of T lymphocytes in the large intestinal tract (**Figures 3C,D, Figures S3C,D**).

We further assessed pro-inflammatory cytokine secretion in intestinal ex vivo biopsies. At day 9 p.i., PLC, but not PACAP treated mice exhibited higher IL-6 concentrations in their ileum as compared to naive counterparts (**Figure 4A**). In addition, TNF secretion was far less pronounced in the ileum and MLN of mice from the PACAP cohort as compared to PLC control animals (**Figures 4B,C**). Hence, PACAP exerts potent inflammationalleviating effects in the intestinal tract of hma mice during subacute ileitis that are not restricted to the terminal ileum.

#### Changes in Gut Microbiota Composition in PACAP Treated Mice With a Human Gut Microbiota Suffering From Subacute Ileitis

Given that intestinal inflammatory conditions are accompanied by shifts in commensal gut microbiota composition of mice

percentiles of the median (black bar inside the boxes). The total range and significance levels determined by one-way ANOVA test followed by Tukey post-correction test for multiple comparisons are shown. Total numbers of analyzed animals are given in parentheses. Data were pooled from four independent experiments.

and men termed dysbiosis (15, 19, 26, 28–30), we quantitatively surveyed the main gut bacterial groups during subacute ileitis development in PACAP and PLC treated mice applying culture-independent 16S rRNA based methods (**Figures 5A-H**). Irrespective of the treatment regimen, the total eubacterial loads in the ileal lumen slightly declined until day 9 p.i. (**Figure 5A**). Conversely, ileitis development was accompanied by higher gene numbers of enterobacteria and enterococci in the ilea of PLC, but not PACAP mice (**Figures 5B,C**), whereas lactobacilli loads were higher in PACAP treated mice at day 9 p.i. as compared to both,

T. gondii infected PLC treated mice and naive control animals (**Figure 5D**). Remarkably, bifidobacteria were only marginally abundant in mice suffering from subacute ileitis (**Figure 5E**), with a trend toward higher loads in PACAP vs. PLC mice at day 9 p.i. (**Figure 5E**). Furthermore, Clostridium coccoides gene numbers were lower in the ileum derived from T. gondii infected mice of either cohort (**Figure 5G**), whereas this was the case for Clostridium leptum in PLC mice (**Figure 5H**), but not PACAP treated counterparts (**Figure 5H**). Hence, subacute ileitis development in hma mice was accompanied with distinct shifts in the microbiota composition of the inflamed ileum, but to a lesser extent upon PACAP treatment.

# Extra-intestinal Anti-inflammatory Effects in PACAP Treated Mice With a Human Gut Microbiota Suffering From Subacute Ileitis

We next addressed whether anti-inflammatory effects upon PACAP treatment of hma mice with subacute ileitis were restricted to the intestinal tract or might also be observed in extra-intestinal including systemic compartments. Nine days following ileitis induction multi-fold increased numbers of apoptotic cell numbers could be observed in the livers derived from PLC and PACAP treated mice as compared to naive animals (**Figure 6A**; **Figure S4A**), but with lower counts in the latter as compared to the former (**Figure 6A**; **Figure S4A**). Irrespective of the treatment regimen, increases in apoptotic hepatic cell numbers were paralleled by more than three-fold higher numbers of T lymphocytes in the livers of T. gondii infected as compared to naive mice (**Figure 6B**; **Figure S4B**). Furthermore, multi-fold elevated numbers of both apoptotic cells and T lymphocytes could be assessed in the lungs during subacute ileitis (**Figures 6C,D**; **Figures S4C,D**), but with lower counts in PACAP as compared to PLC treated mice at day 9 p.i. (**Figures 6C,D**; **Figures S4C,D**).

We next measured pro-inflammatory cytokine secretion in ex vivo biopsies derived from respective extra-intestinal compartments and detected less distinctly increased INF-γ concentrations in the liver and lungs of PACAP as compared to PLC treated mice at day 9 p.i. (**Figure 7**). As for the ileum, subacute ileitis induction was further accompanied by elevated systemic concentrations of pro-inflammatory cytokines such as TNF and IL-6 (**Figure 8**). Strikingly, PACAP treatment resulted in ∼50% lower TNF and IL-6 concentrations measured in serum samples taken at day 9 p.i. as compared to PLC control mice (**Figure 8**). Hence, the profound anti-inflammatory effects exerted by PACAP treatment of hma mice during subacute ileitis were not restricted to the intestinal tract, but could also be observed in extra-intestinal and even systemic compartments.

# DISCUSSION

Several in vitro and in vivo studies revealed that PACAP exerts its neuroprotective properties via immune-modulatory and anti-apoptotic mechanisms (5, 6). Given its virtual ubiquitous expression, however, PACAP rather acts as a pleiotropic immune regulator and hence, also beyond the nervous system (6, 31). In fact, our previous work revealed that synthetic PACAP application starting prior acute ileitis induction (i.e., prophylactic regimen) ameliorated intestinal as well as extraintestinal sequelae of peroral high-dose T. gondii infection in a time-of-treatment dependent manner (17) that is characterized by a T cell-driven pro-inflammatory cytokine storm with fatal outcome within 1 week (14, 15). In the present preclinical intervention study we provide additional insights into the inflammation-ameliorating properties of exogenous PACAP. The here applied subacute infection model following peroral lowdose T. gondii infection of mice with a human gut microbiota has been established by our group very recently (20) and is

quantitative Real-Time PCR amplifying bacterial 16S rRNA variable regions of the main intestinal bacterial groups (expressed as 16S rRNA gene numbers per ng DNA) including the total eubacterial load as indicated. Box plots represent the 75 and 25th percentiles of the median (black bar inside the boxes). The total range and significance levels determined by one-way ANOVA test followed by Tukey post-correction test for multiple comparisons are shown. Total numbers of analyzed animals are given in parentheses. Data were pooled from four independent experiments.

characterized by a non-lethal, far less acute course of small intestinal inflammation (as compared to high-dose T. gondii infection), and importantly, mimics human gut microbiota conditions. The low-dose T. gondii infection model of hma mice thus provides valuable opportunities to further dissect the molecular mechanisms underlying the interactions between pathogen, host immunity, and human gut microbiota during small intestinal inflammation (20).

In our present work we demonstrate that PACAP applied in a therapeutic regimen (starting post ileitis induction) exerts anti-inflammatory effects during subacute ileitis of (with respect to their gut microbiota) "humanized" mice as indicated by (1) better clinical / macroscopic conditions (including lack of body weight loss and of shrinkage of the intestinal lengths during ileitis development), (2) less distinct histopathological changes in the terminal ileum, (3) lower numbers of apoptotic epithelial cells and of T lymphocytes in both, ileum and colon, (4) less intestinal secretion of pro-inflammatory cytokines such as TNF and IL-6, (5) less apoptosis in extra-intestinal organs such as liver and lungs that is accompanied by (6) lower pulmonal T cell numbers, and (7) less IFN-γ secretion in liver and lungs, and, remarkably, (8) lower systemic concentrations of pro-inflammatory cytokines such as TNF and IL-6. Furthermore, (9) inflammation-associated gut microbiota shifts were less pronounced following PACAP as compared to PLC treatment.

The potent anti-inflammatory effects of exogenous PACAP within the intestinal tract as assessed in our actual and previous (17) study are further supported by two previous reports demonstrating that PACAP−/− mice were suffering from more severe DSS-induced colitis as compared to wildtype counterparts (9, 10). When synthetic PACAP was applied via the intraperitoneal route, however, the inflammatory phenotype could be alleviated (9).

Even though peroral T. gondii infection of susceptible mice is known to primarily affect the terminal ileum, we extended our focus to the large intestines here. We did indeed observe marked T. gondii induced colonic inflammatory responses as indicated by multi-fold increased numbers of apoptotic

FIGURE 6 | Apoptotic epithelial cell and T lymphocyte responses in extra-intestinal compartments following PACAP treatment of mice with a human gut microbiota suffering from subacute ileitis. Subacute ileitis was induced by T. gondii infection of mice harboring a human gut microbiota (day 0). Starting 3 days post-infection (p.i.), mice were either treated with PACAP or placebo (PLC). Uninfected mice with a human microbiota served as control animals (Naive). At day 9 p.i., the average numbers of apoptotic epithelial cells [Casp3+; (A,C)] and of T lymphocytes [CD3+; (B,D)] in at least six high power fields (HPF) were quantitatively assessed in paraffin sections of ex vivo biopsies derived from liver (A,B) and lung (C,D) applying in situ immunhistochemistry. Box plots represent the 75 and 25th percentiles of the median (black bar inside the boxes). The total range and significance levels determined by one-way ANOVA test followed by Tukey post-correction test for multiple comparisons are shown. Total numbers of analyzed animals are given in parentheses. Data were pooled from four independent experiments.

colonic epithelial cells that could be effectively lowered by therapeutic PACAP application, which also held true for T lymphocytes within the colonic mucosa and lamina propria. In previous in vitro studies PACAP was shown to inhibit proliferation and migration of T lymphocytes and associated pro-inflammatory cytokine release (3, 32). An in vivo study further revealed that PACAP treated mice suffering from acute peritonitis displayed a diminished influx of lymphocytes into the peritoneal cavity (33).

Interestingly, synthetic PACAP was not sufficient to lower T. gondii induced colonic pro-inflammatory cytokine secretion (not shown), whereas this was the case in ex vivo biopsies

FIGURE 7 | Extra-intestinal pro-inflammatory cytokine responses following PACAP treatment of mice with a human gut microbiota suffering from subacute ileitis. Subacute ileitis was induced by T. gondii infection of mice harboring a human gut microbiota (day 0). Starting 3 days post-infection (p.i.), mice were either treated with PACAP or placebo (PLC). Uninfected mice with a human microbiota served as control animals (Naive). At day 9 p.i., IFN-γ secretion was assessed in ex vivo biopsies derived from the liver (A) and from lung (B). Box plots represent the 75 and 25th percentiles of the median (black bar inside the boxes). The total range and significance levels determined by one-way ANOVA test followed by Tukey post-correction test for multiple comparisons are shown. Total numbers of analyzed animals are given in parentheses. Data were pooled from four independent experiments.

FIGURE 8 | Systemic pro-inflammatory cytokine responses following PACAP treatment of mice with a human gut microbiota suffering from subacute ileitis. Subacute ileitis was induced by T. gondii infection of mice harboring a human gut microbiota (day 0). Starting 3 days post-infection (p.i.), mice were either treated with PACAP or placebo (PLC). Uninfected mice with a human microbiota served as control animals (Naive). At day 9 p.i., secretion of pro-inflammatory cytokines such as TNF (A) and IL-6 (B) were measured in serum samples. Box plots represent the 75 and 25th percentiles of the median (black bar inside the boxes). The total range and significance levels determined by one-way ANOVA test followed by Tukey post-correction test for multiple comparisons are shown. Total numbers of analyzed animals are given in parentheses. Data were pooled from four independent experiments.

derived from the ileum and from MLN. Our present observations are supported by our previous reports in both acute (25, 26) and subacute (20) murine ileitis further emphasizing that T. gondii induced immunopathology does not exclusively affect the terminal ileum, but might also affect the large intestines.

The well-known anti-apoptotic properties of PACAP were not restricted to the intestinal tract, but could also be assessed in extra-intestinal organs such as the liver and lungs and were paralleled by decreased IFN-γ secretion in respective organs. In line, PACAP could exert potent protective effects in human hepatocytes in vitro and in a murine model of liver ischemia and oxidative stress (34, 35) as well as in endotoxin-induced acute lung inflammation (36). Notably, synthetic PACAP analogs have been developed for the treatment of bronchial asthma given the anti-inflammatory and broncho-relaxant properties of the neuropeptide (36, 37).

We further addressed whether the observed inflammationalleviating responses in PACAP treated mice were paralleled by less translocation of viable bacteria originating from the commensal gut microbiota to extra-intestinal including systemic compartments due to less distinct epithelial barrier damage. It is well-known that during acute ileitis bacterial commensals including Escherichia coli overgrowing the ileal lumen might migrate through the compromised intestinal epithelial barrier (i.e., "leaky gut"), come in close contact to immune cells residing in the lamina propria which might subsequently result in an exacerbation of the inflammatory immune responses (38–40). Interestingly, in our actual study bacteria could neither be cultured from MLN, nor from extra-intestinal compartments including liver, lungs and cardiac blood. Our recent study, however, revealed that mean commensal bacterial translocation rates of more than 80% could be assessed in liver and lungs during lethal acute ileitis (25, 26).

One might argue that the better outcome observed in PACAP treated hma mice with T. gondii induced ileitis was due to direct anti-microbial effects of the applied synthetic compound, given that both anti-parasitic (directed against Trypanosoma brucei) (41) and anti-bacterial (42) effects have been reported recently. In both our previous and actual studies we could, however, exclude any antimicrobial effects of the working solutions containing synthetic PACAP in vitro (17). Furthermore, the start of exogenous PACAP 3 days after T. gondii infection, in addition to comparable parasitic DNA loads as assessed in ileal ex vivo biopsies argue against a biological relevant anti-microbial effect of PACAP.

Meanwhile it is well-established that the orchestrated mutualistic microbiota-host relationships are of utmost importance for host cell physiology, immune homeostasis and resistance to disease (21, 43). Perturbations within the complex microbial ecosystem in the gastrointestinal tract are associated with increased susceptibility of the host to distinct intestinal morbidities including IBD, irritable bowel syndrome and coeliac disease (21, 44, 45). Likewise, intestinal inflammatory conditions are paralleled by shifts in the intestinal microbiota composition (15, 25, 26, 28–30, 46, 47). This phenomenon could also be observed in our actual survey of the microbiota composition within the inflamed ileal lumen, given that subacute ileitis development was associated with increases in enterobacteria (including E. coli) and enterococci, whereas obligate anaerobic Clostridium coccoides and Clostridium leptum gene numbers decreased until day 9 p.i. These microbiota shifts are supported by our previous results obtained from lethal acute ileitis of mice, but with a conventional (i.e., murine) gut microbiota (15, 46, 47). Remarkably, neither shifts toward increased enterobacteria and enterococci, nor to decreased numbers of Clostridium leptum could be assessed in the ilea of PACAP treated mice. Furthermore, PACAP treatment was associated with higher abundances of (potentially probiotic) lactobacilli that had been reduced during acute ileitis (15).

Interestingly, PACAP could inhibit TLR-4 activation in a model of traumatic brain injury (48). Given that T. gondii induced ileitis is initiated and further worsened by TLR-4 dependent signaling of bacterial LPS originating from the cell walls of Gram-negative commensals including enterobacteria such as E. coli accumulating in the inflamed ileal lumen (46, 47), alleviation of the TLR-4 dependent scenario constitutes a mechanistic corner stone of the multi-facetted "health-beneficial actions" of PACAP treatment.

Strikingly, exogenous PACAP could not only sufficiently dampen ileal, but also serum TNF and IL-6 concentrations, pointing toward potent systemic anti-inflammatory properties of the synthetic compound, which could also be observed in lethal acute ileitis (17). These systemic effects of exogenous PACAP are supported by several studies where PACAP could efficiently prevent from experimental endotoxin sepsis and shock (49–51).

Given the time-of-treatment dependent anti-inflammatory effect of exogenous PACAP observed during acute ileitis (17), one might further argue that starting PACAP application to hma mice before subacute ileitis induction (i.e., prophylactic regimen) might yield even more pronounced anti-apoptotic and anti-inflammatory effects in intestinal and extra-intestinal including systemic compartments. This should be addressed in future studies.

In summary, our preclinical intervention study provides strong evidence that synthetic PACAP alleviates subacute ileitis and extra-intestinal including systemic sequelae of T cell-driven immunopathology. We conclude that synthetic PACAP might open novel options for the (adjunct) therapy and/or prophylaxis of intestinal inflammation including IBD and further supports the pathophysiological role of the gutbrain axis.

#### ETHICS STATEMENT