Lactobacillus johnsonii ameliorates intestinal, extra-intestinal and systemic pro-inflammatory immune responses following murine Campylobacter jejuni infection

Abstract

Campylobacter jejuni infections are progressively increasing worldwide. Probiotic treatment might open novel therapeutic or even prophylactic approaches to combat campylobacteriosis. In the present study secondary abiotic mice were generated by broad-spectrum antibiotic treatment and perorally reassociated with a commensal murine Lactobacillus johnsonii strain either 14 days before (i.e. prophylactic regimen) or 7 days after (i.e. therapeutic regimen) peroral C. jejuni strain 81-176 infection. Following peroral reassociation both C. jejuni and L. johnsonii were able to stably colonize the murine intestinal tract. Neither therapeutic nor prophylactic L. johnsonii application, however, could decrease intestinal C. jejuni burdens. Notably, C. jejuni induced colonic apoptosis could be ameliorated by prophylactic L. johnsonii treatment, whereas co-administration of L. johnsonii impacted adaptive (i.e. T and B lymphocytes, regulatory T cells), but not innate (i.e. macrophages and monocytes) immune cell responses in the intestinal tract. Strikingly, C. jejuni induced intestinal, extra-intestinal and systemic secretion of pro-inflammatory mediators (such as IL-6, MCP-1, TNF and nitric oxide) could be alleviated by peroral L. johnsonii challenge. In conclusion, immunomodulatory probiotic species might offer valuable strategies for prophylaxis and/or treatment of C. jejuni induced intestinal, extra-intestinal as well as systemic pro-inflammatory immune responses in vivo.

Figure 1

Kinetics of intestinal L. johnsonii and/or C. jejuni strain 81–176 loads in perorally reassociated secondary abiotic mice. Secondary abiotic mice were generated by broad-spectrum antibiotic treatment and perorally reassociated with (a) L. johnsonii (Lac; white squares) on day (d) −14, (b) C. jejuni strain 81–176 (Cj; black circles) on d0, (c) L. johnsonii on day −14 and C. jejuni strain 81–176 on d0, or (d) C. jejuni strain 81–176 (d-7) and L. johnsonii on d0 as described in methods. Bacterial colonization densities were assessed in fecal samples (CFU/g, colony forming units per gram) over time upon reassociation as indicated by culture. Medians (black bars) and levels of significance (p-value) determined by Mann-Whitney U test are indicated. Numbers of analyzed mice are given in parentheses. Data were pooled from three independent experiments. N.d.: not determined.

Figure 2

Apoptotic and proliferating cells in the colonic epithelium of C. jejuni strain 81–176 and/or L. johnsonii associated secondary abiotic mice. Secondary abiotic mice were perorally infected with C. jejuni strain 81–176 (Cj) and associated with L. johnsonii (Lac) either 14 days before (prophylactic regimen, Proph; white squares) or 7 days thereafter (therapeutic regimen, Therap; black squares) and compared to mono-associated mice (+Lac, white circles; +Cj, black circles). The average number of colonic (a) apoptotic cells (positive for caspase-3, Casp3) and (b) proliferating cells (positive for Ki67) from six high power fields (HPF, 400x magnification) per animal was determined microscopically in immunohistochemically stained colonic paraffin sections at days 21 or 28 following initial C. jejuni or L. johnsonii infection, respectively. Naive mice (black diamonds) served as uninfected controls. Medians (black bars), levels of significance (p-values) determined by one-way ANOVA test followed by Tukey post-correction test for multiple comparisons and numbers of analyzed animals (in parentheses) are indicated. Data were pooled from three independent experiments.

Figure 3

Colonic immune cell responses in C. jejuni strain 81–176 and/or L. johnsonii reassociated secondary abiotic mice. Secondary abiotic mice were perorally infected with C. jejuni strain 81–176 (Cj) and associated with L. johnsonii (Lac) either 14 days before (prophylactic regimen, Proph; white squares) or 7 days thereafter (therapeutic regimen, Therap; black squares) and compared to mono-associated mice (+Lac, white circles; +Cj, black circles). The average number of colonic (a) T lymphocytes (positive for CD3), (b) regulatory T cells (Treg; positive for FOXP3), (c) B lymphocytes (positive for B220), and (d) macrophages and monocytes (positive for F4/80) from six high power fields (HPF, 400x magnification) per animal was determined microscopically in immunohistochemically stained colonic paraffin sections at days 21 or 28 following initial C. jejuni or L. johnsonii infection, respectively. Naive mice (black diamonds) served as uninfected controls. Medians (black bars), levels of significance (p-values) determined by one-way ANOVA test followed by Tukey post-correction test for multiple comparisons and numbers of analyzed animals (in parentheses) are indicated. Data were pooled from three independent experiments.

Figure 4

Intesinal secretion of pro- and anti-inflammatory mediators in C. jejuni strain 81–176 and/or L. johnsonii reassociated secondary abiotic mice. Secondary abiotic mice were perorally infected with C. jejuni strain 81–176 (Cj) and associated with L. johnsonii (Lac) either 14 days before (prophylactic regimen, Proph; white squares) or 7 days thereafter (therapeutic regimen, Therap; black squares) and compared to mono-associated mice (+Lac, white circles; +Cj, black circles). Colonic (a) IL-6, (b) MCP-1 and (c) IL-10 concentrations as well as ileal (d) TNF, (e) nitric oxide and (f) IL-10 secretion were determined in ex vivo biopsies derived at days 21 or 28 following initial C. jejuni or L. johnsonii infection, respectively. Naive (N) mice (black diamonds) served as uninfected controls. Medians (black bars), levels of significance (p-value) determined by one-way ANOVA test followed by Tukey post-correction test for multiple comparisons and numbers of analyzed animals (in parentheses) are indicated. Data were pooled from three independent experiments.

Figure 5

Secretion of pro- and anti-inflammatory mediators in mesenteric lymph nodes of C. jejuni strain 81–176 and/or L. johnsonii reassociated secondary abiotic mice. Secondary abiotic mice were perorally infected with C. jejuni strain 81–176 (Cj) and associated with L. johnsonii (Lac) either 14 days before (prophylactic regimen, Proph; white squares) or 7 days thereafter (therapeutic regimen, Therap; black squares) and compared to mono-associated mice (+Lac, white circles; +Cj, black circles). (a) Nitric oxide and (b) IL-10 concentrations were determined in ex vivo biopsies derived from mesenteric lymph nodes (MLN) at days 21 or 28 following initial C. jejuni or L. johnsonii infection, respectively. Naive (N) mice (black diamonds) served as uninfected controls. Medians (black bars), level of significance (p-value) determined by one-way ANOVA test followed by Tukey post-correction test for multiple comparisons and numbers of analyzed animals (in parentheses) are indicated. Data were pooled from three independent experiments.

Figure 6

Secretion of pro- and anti-inflammatory mediators in livers of C. jejuni strain 81–176 and/or L. johnsonii reassociated secondary abiotic mice. Secondary abiotic mice were perorally infected with C. jejuni strain 81–176 (Cj) and associated with L. johnsonii (Lac) either 14 days before (prophylactic regimen, Proph; white squares) or 7 days thereafter (therapeutic regimen, Therap; black squares) and compared to mono-associated mice (+Lac, white circles; +Cj, black circles). (a) TNF, (b) IL-6 and (c) IL-10 concentrations were determined in ex vivo biopsies derived from livers at days 21 or 28 following initial C. jejuni or L. johnsonii infection, respectively. Naive (N) mice (black diamonds) served as uninfected controls. Medians (black bars), levels of significance (p-value) determined by one-way ANOVA test followed by Tukey post-correction test for multiple comparisons and numbers of analyzed animals (in parentheses) are indicated. Data were pooled from three independent experiments.

Figure 7

Splenic secretion of pro- and anti-inflammatory mediators of C. jejuni strain 81–176 and/or L. johnsonii reassociated secondary abiotic mice. Secondary abiotic mice were perorally infected with C. jejuni strain 81–176 (Cj) and associated with L. johnsonii (Lac) either 14 days before (prophylactic regimen, Proph; white squares) or 7 days thereafter (therapeutic regimen, Therap; black squares) and compared to mono-associated mice (+Lac, white circles; +Cj, black circles). (a) TNF, (b) INF-γ, (c) MCP-1, (d) IL-6, and (e) IL-10 concentrations were determined in ex vivo biopsies derived from spleens at days 21 or 28 following initial C. jejuni or L. johnsonii infection, respectively. Naive (N) mice (black diamonds) served as uninfected controls. Medians (black bars), levels of significance (p-value) determined by one-way ANOVA test followed by Tukey post-correction test for multiple comparisons and numbers of analyzed animals (in parentheses) are indicated. Data were pooled from three independent experiments.

Figure 8

Pro-inflammatory cytokine secretion in serum of C. jejuni strain 81–176 and/or L. johnsonii reassociated secondary abiotic mice. Secondary abiotic mice were perorally infected with C. jejuni strain 81–176 (Cj) and associated with L. johnsonii (Lac) either 14 days before (prophylactic regimen, Proph; white squares) or 7 days thereafter (therapeutic regimen, Therap; black squares) and compared to mono-associated mice (+Lac, white circles; +Cj, black circles). (a) TNF, (b) MCP-1 and (c) IL-6 concentrations were determined in serum samples at days 21 or 28 following initial C. jejuni or L. johnsonii infection, respectively. Naive (N) mice (black diamonds) served as uninfected controls. Medians (black bars), levels of significance (p-value) determined by one-way ANOVA test followed by Tukey post-correction test for multiple comparisons and numbers of analyzed animals (in parentheses) are indicated. Data were pooled from three independent experiments.