Intranasal bacteria induce Th1 but not Treg or Th2

Abstract

Commensal microorganisms colonize the nasal mucosa without inducing inflammation. Pathogens perturbing the commensal flora often invade evading immune defenses. The different types of adaptive responses that drive the distinct behaviors of commensals and pathogens, allowing one to persist at mucosal surfaces and the other to survive within tissues, are not yet clear. In the present work we demonstrate that although crossing epithelial barriers, the commensal Lactobacillus murinus stimulates epitope-specific CD4(+) T cells in nasal-associated lymphoid tissue (NALT) less efficiently than the pathogen Streptococcus pyogenes. In NALT antigen-presenting cells other than CCR6(+) CD11c(+) dendritic cells process and present the microbial antigens. Effector/memory CD4(+) T cells generated after intranasal priming with L. murinus and S. pyogenes surprisingly express similar proinflammatory cytokines and are not CD25+/FoxP3+ T-regulatory cells when recirculating in the spleen. These findings suggest that when a commensal crosses the nasal epithelial barrier it induces a proinflammatory response similar to a pathogen but without causing disease.

Figure 1

L. murinus and S. pyogenes are recovered from nasal-associated lymphoid tissue (NALT) tissues. (a) Summary of experimental design. (b) Bacterial recovery from NALT tissues after anesthetized mice were inoculated intranasally once with 2×10⁸ CFU of OVA⁺ S. pyogenes or with 1×10⁹ CFU of OVA⁺ L. murinus for 3 consecutive days in two aliquots of 7.5 µl each in phosphate-buffered saline (PBS) delivered at in front of each nostril. At 4 days after S. pyogenes and 24 h after L. murinus last inoculations NALT tissues were dissected, weighed, rinsed in PBS, mashed, and plated in serial dilutions on Lactobacilli deMan–Rogosa–Sharpe (MRS) agar or blood–Todd–Hewitt yeast extract (THY) agar plates containing 5 µg/ml erythromycin for 24 h at 37°C.

Figure 2

T cells are located in the T-cell rich region and B cells are facing the nasal-associated follicular epithelium (NFAE). Nasal-associated lymphoid tissue (NALT) of a BALB/c mouse 4 days after intranasal infection with OVA⁺ S. pyogenes. Immunohistology at ×20 magnification of NALT tissues double stained with Cy3-amplified (a) IgG2a isotypes or (b, c) anti-OVA T-cell receptor (TCR) (KJ1-26 mAb) and fluorescein isothiocyanate (FITC)-conjugated anti-B220 and recolored in Adobe Photoshop.

Figure 3

CD11c cells localize under the epithelium of nasal-associated lymphoid tissue (NALT) 4 days after bacterial inoculation. Immunohistology at ×20 magnification of triple-stained NALT tissue oriented with the luminal side is on the right side of the images. The three columns display mice treated with phosphate-buffered saline (PBS) (a, d, g), OVA⁺ L. murinus (b, e, h), or S. pyogenes (c, f, i). The first row displays NALT tissues stained with IgG1 and IgG2a isotypes and anti-B220 as shown in a and b or polyclonal rabbit IgG and anti-B220 as shown in c. The tissues in the second row as shown in d–f were stained with anti-CD11c, KJ1-26, or anti B220 displaying B-cell follicles (blue), OVA-specific T cells (green), and CD11c⁺ cells (red). Images in the third row display tissues double stained with rabbit IgG anti-mouse CCR6 and anti-B220 as shown in g–i. Mice were inoculated intranasally once with 2×10⁸ CFU of OVA⁺ S. pyogenes or with 1×10⁹ CFU of OVA⁺ L. murinus for 3 consecutive days in two aliquots of 7.5 µl each in PBS delivered at in front of each nostril. At 4 days after S. pyogenes and 24 h after L. murinus last inoculations the maxillae containing NALT tissues were dissected, demineralized, and embedded in OCT for cryosection.

Figure 4

Direct visualization of proliferating T cells in nasal-associated lymphoid tissue (NALT) by immunohistology 4 days after bacterial inoculation. (a) Histogram estimating the fraction of activated antigen-specific T cells when normalized against the background phosphate-buffered saline (PBS) group 4 days after initial priming. The percentage of dividing cells is estimated by calculating the number of yellow pixels (CFSE^high (green) and KJ1-26⁺ cells (red)) minus the number of red pixels (all KJ1-26⁺ cells) pixels divided by the total number of red pixels measured from mice inoculated with (b) PBS, (c) OVA⁺ L. murinus, or (d) OVA⁺ S. pyogenes. 5-(and-6)-Carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled cells (green channel) that are also KJ1-26⁺ (red dots) will appear yellow after merging of the colors. Cells that divide lose their CFSE and become red because they are now stained only with the KJ1-26 mAb.

Figure 5

L. murinus primes OVA-specific T cells less efficiently than S. pyogenes. (A) Flow cytometry example of contour plots of splenocytes from mice inoculated intranasally with L. murinus for 9 days. (a) R1 identifies lymphocytes in forward and side scatter plot. (b) Subgate of R1 identifies CD4⁺ leukocytes (R2). (c) Subgate of R2 excludes B cells, CD8 T cells, CD11c⁺ CD4⁺ DCs, and potential circulating double-positive lymphocytes. (d) Subgate of R3 identifies the percentage of OVA-specific T cells (R4) out of total lymphocytes (R1). (e) Subgate of R4 identifies the percentage of dividing OVA-specific T cells when the ratio of cells diluting their 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) in the M2/M1 gates×100 is calculated. (B, C) Percentage of dividing CD4⁺ KJ1-26⁺ T cells in spleen 21 days after initial intranasal inoculation with phosphate-buffered saline (PBS), 2×10⁹ OVA⁺ L. murinu s or 2×10⁸ OVA⁺ S. pyogenes for 2 days (gray bars) or 14 days (white bars). To avoid septicemia, S. pyogenes was heat killed at 65 °C for 1 h before the inoculation for 14 days. Data shown are averages±s.e.m. from three experiments (n = 6 mice per data point). T-cell proliferation was significantly greater (P < 0.05) in each treatment group when compared to PBS control (* P < 0.05; Student’s t-test).

Figure 6

Nasal inoculum of L. murinus and S. pyogenes induce proliferation first in nasal-associated lymphoid tissue (NALT) and cervical lymph nodes. (a) Summary data showing the percentage of dividing CD4⁺ KJ1-26⁺ T cells in NALT and cervical LN at day 9 after initial intranasal inoculation with phosphate-buffered saline (PBS), 10–15 µg of OVA 323–339 peptide (pOVA), single inoculation of OVA⁺ S. pyogenes or daily inoculation of OVA⁻ or OVA⁺ L. murinus for 9 consecutive days. Data shown are averages±s.e.m. from three experiments (n = 6 mice per data point). (b) Representative flow cytometry histograms showing CFSE dilution profiles and percentage dividing CD4⁺ KJ1-26⁺ T cells isolated from NALT of mice 9 days after inoculation with antigens. (c) Summary data from from experiments showing the percentage of divided CD4⁺ KJ1-26⁺ T cells in the spleen 14 and 21 days after initial intranasal inoculation with PBS, single inoculation of OVA⁺ S. pyogenes, or daily inoculation of OVA⁺ L. murinus for 14 consecutive days or single intravenous inoculation of 100 µg of OVA peptide and 50 µg E. coli LPS. Data shown are averages±s.e.m. from four experiments (n = 8 mice per data point). * = Student’s t-test P < 0.05 when compared to PBS group.

Figure 7

L. murinus or S. pyogenes do not induce the maturation central T-regulatory (Treg) cells. (A) Using the gating strategy outlined in Figure 5A, the fraction of naive OVA-specific Treg cells that is generated 21 days after adoptive transfer disappears after priming with L. murinus or S. pyogenes. Data represent averages of five experiments; total mice per treatment group n = 10. (B) Representative contour plots from six-color flow cytometry indicating the expression of FoxP3 (a–d) and CD25 (e–h) of cells labeled with CFSE stained intracellularly with anti-IL-2 and anti-CD25 mAbs (j–l). Panels “a–d” and “e–l” are representative of two independent experiments.

Figure 8

Proinflammatory cytokines are expressed and released in effector/memory antigen-specific T cell after rechallege in vivo with OVA peptide. (A) Using the gating strategy outlined in Figure 5A we report contour plots of splenocytes of T cell adoptively transferred mice 18–21 days after initial priming and 2.5 h after OVA IV peptide restimulation. Cells were permeabilized and stained for intracellular cytokines interleukin-2 (IL-2) and tumor-necrosis factor-α (TNFα). Mice were primed intranasally with phosphate-buffered saline (PBS) (a, e), daily inoculations of OVA⁺ L. murinus for 14 days (b, f), single inoculation of OVA⁺ S. pyogenes (c, g), or intravenous inoculation of OVA peptide plus adjuvant E. coli LPS (d, h). (B) Flow cytometry contour plots of spleens of T cell adoptively transferred mice 21 days earlier and primed intranasally as described in A. Mice were restimulated with OVA peptide 100 µg intravenously. Splenocytes were harvested 2.5 h later, incubated with cytokine-capture mAb (Miltenyi Biotech) for 60 min secretion period at 37°C. Cytokines were revealed with PE-labeled anticytokine sandwich mAb and analyzed in flow cytometry. Priming with OVA⁺ L. murinus for 14 days induces T cells to release of interferon (IFN)γ and IL-2 (a′, b′ upper left quadrants) but not of IL-4 or IL-10 (c′, d′ upper left quadrants) after 2.5 h rechallenge with OVA peptide. Single inoculation of OVA⁺ S. pyogenes parallels the IFNγ and IL-2 (e′, f′ upper left quadrants) and IL-4 and IL-10 release (g′, h′ upper left quadrants).