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Published ahead of print on September 3, 2004, doi:10.1165/rcmb.2004-0207OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 31, pp. 657-662, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2004-0207OC

Immune Response to Mycoplasma pulmonis in Nasal Mucosa Is Modulated by the Normal Microbiota

Gert Henriksson, Lars Helgeland, Tore Midtvedt, Pontus Stierna and Per Brandtzaeg

Departments of Clinical Sciences and of Otorhinolaryngology, Karolinska Institute, Huddinge University Hospital, Huddinge, Sweden; Laboratory for Immunohistochemistry and Immunopathology (LIIPAT), Institute of Pathology, Rikshospitalet University Hospital, University of Oslo, Oslo, Norway; and Department of Medical Microbial Ecology, Karolinska Institute, Stockholm, Sweden

Address correspondence to: Prof. Per Brandtzaeg, Institute of Pathology, Rikshospitalet University Hospital, N-0027 Oslo, Norway. E-mail: per.brandtzaeg{at}medisin.uio.no


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The impact of commensal bacteria on lymphocyte responses in the upper airways was studied in rat nasal mucosa after infection with the pathogen Mycoplasma pulmonis. Phenotyping was performed in situ by paired immunofluorescence staining in germ-free (GF) and conventional (CV) rats before and 3 wk after the monoinfection. Intraepithelial lymphocytes had expanded significantly in GF (P = 0.02) but not in CV rats. Furthermore, a striking proportional increase of T-cell receptor (TCR){alpha}ß+CD4+ cells was observed both in the lamina propria and epithelium of GF (P < 0.01) but not of CV rats. Notably, in contrast to the pre-infection state, both mucosal compartments showed a percentage of TCR{alpha}ß+CD4+ cells that was significantly higher in GF (P = 0.03–P < 0.01) than in CV rats after the monoinfection. In parallel, both compartments displayed a percentage of TCR{alpha}ß+ CD8+ cells that was decreased in GF (P < 0.01) but not in CV rats. The small fraction of TCR{gamma}{delta}+ T cells observed (< 5%) did not change quantitatively or phenotypically after infection. The size of organized nose-associated lymphoid tissue was, on average, increased 5.2-fold in GF rats versus 2.6-fold in CV rats. Collectively, our results demonstrated that the normal microbiota modulated markedly the nasal immune response elicited by monoinfection with M. pulmonis.

Abbreviations: antigen-presenting cell, APC • conventional, CV • dendritic cell, DC • germ free, GF • intraepithelial lymphocyte, IEL • lamina propria lymphocyte, LPL • monoclonal antibodies, mAb • nose-associated lymphoid tissue, NALT • natural killer, NK • pattern recognition receptor, PRR • T-cell receptor, TCR • regulatory T cells, Treg cells


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
T lymphocytes play an important role in the regulation of mucosal immune responses to exogenous antigens, but virtually nothing is known about the effect of the indigenous microbiota on such responses in the upper airways (1). Only a few studies of the immunohistology of human nasal mucosa, with or without inflammatory conditions, have been performed (2–4). Conversely, there are several reports documenting that commensal bacteria modulate mucosal immune responses in the gut (reviewed in Refs. 5–7). Germ-free (GF) animals (pathogen-free with no indigenous flora) are known to possess an immature immune system and have been used to study the impact of the normal microbiota on mucosal immunity. Thus, in GF mice and rats, the absence of the stimulation exerted by commensal bacteria affects both the numbers and phenotypes of intestinal T lymphocytes (8, 9) as well as the development of gut-associated lymphoid tissue with its antigen-sampling M cells (10). Importantly, GF mice are defective with regard to induction and maintenance of oral tolerance and also recovery of oral tolerance after its abrogation by bacterial toxins (5–7, 11). It remains unknown whether this positive effect of the commensal flora is mediated through modulation of costimulatory molecules on antigen-presenting cells (APCs) or an effect on intestinal permeability. Notably, indigenous bacteria are important both to establish (12) and to regulate (13) an appropriate epithelial barrier function.

Ichimya and colleagues (14) showed that fewer immunoglobulin (Ig)M+ B cells and CD4+ T cells were present in the upper respiratory tract of GF mice compared with specific pathogen-free conventional (CV) mice. IgG+ and IgA+ B cells, as well as CD8+ T cells, were rare and apparently unchanged. It was concluded that both B and T cells were attracted to the nasal mucosa in response to microbial stimuli from the commensal flora (14). In the rodent nasal cavity, lymphocytes are both diffusely distributed in the mucosa and aggregated in the paired lymphoid organs called nose-associated lymphoid tissue (NALT) (15), which shows similarities to the ileal Peyer's patches but differs with regard to organogenesis. Thus, Peyer's patches consist of organized T- and B-cell compartments at birth, although the development of germinal centers only takes place postnatally under the influence of the microflora. Conversely, NALT anlagen cannot be detected in mice until shortly after birth (16), and germinal center formation is again dependent on exogenous stimuli (17). Functionally, rodent NALT may be equivalent to nasopharyngeal lymphoid tissue called Waldeyer's ring in humans, including the palatine tonsils and adenoids (18, 19), and human nasal mucosa may in addition contain a few isolated (solitary) lymphoid follicles (20). The CD4:CD8 T-cell ratio in rodent NALT has been calculated to be 2.4, apparently having a larger T:B lymphocyte ratio than human Peyer's patches (18).

Mycoplasma pulmonis is an extracellular bacterial pathogen that attaches to rodent respiratory surface epithelium and, over time, causes a chronic mucosal inflammatory condition (21–23). Mycoplasmas induce specific humoral and cell-mediated immunity, as well as nonspecific stimulatory and suppressive effects on the immune system (21). In the interactions between the bacteria and the host cells, some of the immunopathologic events are more damaging than the direct toxic effects of the infecting organism. In the lower respiratory tract of CV rats, M. pulmonis infection has been shown to alter significantly lymphocyte populations in terms of numbers as well as subset distribution (24). The morphologic and microbiological features of the nasal cavity of GF rats related to these pathogens have been described as "chronic respiratory disease" (23).

The aim of this work was to investigate the possible impact that the normal microbiota exerts on the nasal T-cell response in rats monoinoculated with M. pulmonis. For reasons explained previously, including the size of the experimental animals, we have found the rat model quite useful for studies of the effect of bacteria on the responsiveness in various mucosal immune compartments (10). Here, we observed numeric and phenotypic T-cell differences between GF and CV rats in both the epithelium and lamina propria of nasal mucosa, in addition to hypertrophy of NALT as a result of dramatic pathogen-induced T- and B-cell expansion. Our results document that the indigenous microbiota exerts a modulating effect on airway immunity to an infectious agent, thus serving to maintain mucosal homeostasis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Inbred GF and CV rats of the same AGUS strain were reared at the Department of Medical Microbial Ecology, Karolinska Institute, Stockholm, Sweden. The GF rats were kept in lightweight stainless-steel isolators and monitored weekly for GF status. The CV rats were kept in a laboratory animal room and checked quarterly by the National Veterinary Institute (Uppsala, Sweden) by serology and culture for absence of pathogens. All animals were fed a steam-sterilized standard rat chow (R36; Lactamin, Vadstena, Sweden) and had free access to water. Artificial light was available between 6:00 A.M. and 6:00 P.M, the temperature and humidity were maintained at 24°C ± 2°C and 55 ± 10%, respectively. Permission for this study was obtained from the ethics committee at Huddinge Hospital.

Mycoplasma Cultivation and Inoculation
M. pulmonis (strain M 61/82), originally isolated from a rat with pneumonia, was obtained from The National Veterinary Institute (Uppsala, Sweden). After the second passage, the bacteria were stored at –70°C. The medium used for mycoplasma cultivation was as described previously (25). Samples for two inoculations were prepared as follows: thawed mycoplasma was cultured in broth for 2 d. CFUs were counted, and the cultures were appropriately diluted in isotonic phosphate-buffered saline (pH 7.5). The first inoculate contained 2.8 x 109 CFU/ml and the second contained 5 x 107 CFU/ml. GF and CV rats received inoculates through the nasal cavity (drop infection) at 8 wk of age and were killed 3 wk later. Age- and sex-matched GF and CV control rats were analyzed in parallel to the inoculated animals. Each test group (n = 5) had a similar distribution of females (weight, 160–180 g) and males (weight, 200–220 g). There was no apparent weight difference between GF and CV rats before or after the experiments.

Sampling from nostrils and pharynx of inoculated rats was performed with cotton wire swabs immersed in 2.7 ml broth (amoxicillin, phosphate-buffered saline, and horse serum) and left for 6 h at room temperature. Portions of this solution (0.01 ml) were then streaked onto agar plates with overlying broth. The plates were incubated for 7 d in air with 5% CO2 at 37°C. Mycoplasma colonies were identified by indirect immunofluorescence with a primary antiserum against M. pulmonis. If colonies were not detected after 1 wk, the plates were incubated for another 1–2 wk, followed by a new identification.

Tissue Processing
Rat skulls were dissected and the facial bone with nasal mucosa removed. The bony structures of the zygoma, maxillary region, and the frontal bone were thinned out with a drill, which was also used to extract the two front teeth, whereas the more posterior teeth were removed together with the tongue. The nasal cavities were filled with OCT compound (Tissue-Tek, Miles Laboratories, Elkhart, IN), and the specimens were immediately snap-frozen in liquid nitrogen. Serial sections were cut at 9 µm in a Leitz 1720 cryostat (Leitz, Wetzlar, Germany) equipped with a Leitz bone knife. The sectioning plane was perpendicular to the long axis, and the level behind the proximal end of alveoli of the frontal teeth was selected for optimal data collection on the NALT structures as well as on the nasal respiratory and olfactory epithelium. Sections were mounted on poly-L-lysine–coated glass slides, dried overnight at room temperature, and fixed in acetone for 10 min. The slides were then wrapped in foil and kept at –20°C until use.

Immunofluorescence Staining
Lymphocyte cell surface markers were visualized in tissue sections by multicolor immunofluorescence, which was based on indirect staining procedures with combinations of primary unlabeled monoclonal antibodies (mAbs) of different murine IgG subclasses (Table 1) and subclass-specific secondary antibody conjugates as detailed previously (8). For analysis of CD4/CD8{alpha} coexpression by subsets with different T-cell receptor (TCR), cryosections were incubated with IgG1 mAb against TCR{alpha}ß (R73) or TCR{gamma}{delta} (V65), together with IgG2a mAb against CD4 (OX35) or CD8{alpha} (G28). Subsequently, bound mAb was identified with appropriately diluted Cy3-conjugated (‘red’) goat anti-mouse IgG1 and biotinylated goat anti-mouse IgG2a (both from Southern Biotechnology, Birmingham, AL) mixed with 20% rat serum to remove cross-reactivity. A final incubation step included Cy2-conjugated (‘green’) streptavidin (Southern Biotechnology). The same procedure was employed when NKR-P1+ cells were analyzed for CD8{alpha} or CD2 coexpression. When anti-CD3 (G4.18, mouse IgG3) was applied in combination with the anti-TCR mAbs (IgG1), the second incubation step included Cy3-conjugated goat anti-mouse IgG3 and biotinylated goat anti-mouse IgG1 (Southern Biotechnology) absorbed with 20% rat serum, followed by Cy2-conjugated streptavidin as described above. In most of the staining experiments, the epithelium was visualized by adding a rabbit antiserum (1/100) to cytokeratin (our laboratory) in the second step, followed by AMCA-conjugated (‘blue’) goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA).


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  		TABLE 1. Mouse monoclonal antibodies used as primary reagents

 
To analyze the distribution of T and B cells in NALT, three cell surface markers were traced simultaneously. The first incubation step included a mixture of anti-CD3, anti-CD4 and anti–B-cell mAbs, followed by a mixture of subclass-specific goat anti-mouse IgG1, IgG2a, and IgG3 conjugated with Cy3, fluorescein isothiocyanate or biotin, respectively (Southern Biotechnology), and finally AMCA-conjugated streptavidin (Vector). The mixture of anti-CD3 and the anti–B-cell mAbs enabled a satisfactory mapping of the distribution of the T and B cells, but CD4 showed faint expression on dendritic cells and macrophages in addition to strong expression on T cells. Negative controls were obtained by substituting the primary reagents with irrelevant isotype- and concentration-matched mAbs. All antibody reagents were applied for 1 h at room temperature.

Microscopy and Cell Counting
The tissue sections were evaluated at x400 magnification in a Nikon microscope (Eclipse E800; Nikon, Tokyo, Japan) equipped with dichroic filters for selective observation of individual cells with regard to fluorescein isothiocyanate and Cy2 (green), Cy3 (red), or AMCA (blue) emission. A charge-coupled device video camera system (C5810; Hamamatsu Photonics, Hamamatsu, Japan) attached to the microscope was used to capture digitalized fields for computerized multicolor images (Foto-station; Interfoto AS, Oslo, Norway).

Lamina propria lymphocytes (LPLs) and intraepithelial lymphocytes (IELs) were recorded based on appropriate cell surface markers, the latter subset being defined as cells with at least half of the surface profile located within the epithelium. The numerical changes of these cell populations were determined in relation to the estimated length of the surface epithelium in the actual section (IELs and LPLs per mm epithelium). Proportions of TCR{alpha}ß+ LPLs were determined by evaluating at least 200 cells for concomitant expression of a subset marker within at least half of the cross-sectional area of the nasal cavity (i.e., covering a region around one NALT structure and the respiratory epithelium of one of the two nasal cavities, including the olfactory epithelium in its superior region). Because IELs were rare, counts from the whole cross-sectional area of the nasal cavity were accumulated, excluding intraepithelial cells of NALT regions. A median of 113 IELs were enumerated (range, 49–189 cells in each specimen).

The size of NALT was estimated by measuring the edges of the organized lymphoid aggregates that formed an approximately rectangular or triangular area, or the combination of a triangle stacked on a rectangle. Lymphoid cells within NALT itself were too densely packed for enumeration.

Statistical Analysis
The Mann-Whitney U-test (two-tailed) was used for the statistical analysis, and P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Nasal T-Cell Phenotypes in Uninfected Conventional Rats
CD3+ LPLs were observed as single scattered cells or in small clusters located mainly in the subepithelial region. CD3+ IELs were distributed throughout the surface epithelium, but occurred most frequently at the base of the nasal cavity. Both LPLs and IELs were scarce in the superior region of the cavities where the olfactory epithelium occurs. Such site-specific differences in the distribution of T lymphocytes and other immune cells have previously been described in the respiratory tract (related to the larynx) of rats (26). Notably, nasal lymphocytes were mainly located adjacent to the NALT structures, and more than half of them were not CD3+ T cells, but rather natural killer (NK) cells (see below, and cell distribution exemplified in Figure 4E).

Most nasal CD3+ LPLs and IELs expressed TCR{alpha}ß (> 90%), and only a few expressed TCR{gamma}{delta} (~ 5%). Surprisingly, most {alpha}ß+ T cells in the surface epithelium were CD4+ (median, 65%), although a substantial fraction expressed CD8 (31%). As expected, most {alpha}ß+ T cells in the lamina propria were CD4+ (63%), whereas the proportion of CD8+ cells (21%) tended to be somewhat lower than in the epithelium (Figures 1 and 2).



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  		Figure 1. The percentage of CD4+ T cells of the total TCR{alpha}ß+ cell population is two tissue compartments of nasal mucosa before and after monoinfection with M. pulmonis in germ-free and conventional rats. Medians and significant differences are indicated. Open circles, uninfected; closed circles, infected, 3 wk.

 


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  		Figure 2. The percentage of CD8+ T cells of the total TCR{alpha}ß+ cell population is two tissue compartments of nasal mucosa before and after monoinfection with M. pulmonis in germ-free and conventional rats. Medians and significant differences are indicated. Open circles, uninfected; closed circles, infected, 3 wk.

 
Nasal T-Cell Phenotypes and NALT Size Are Influenced by the Normal Microbiota
The number of TCR{alpha}ß+ LPLs in nasal mucosa estimated per millimeter of surface epithelium was quite similar in GF and CV rats, whereas the corresponding IEL subset was more numerous in the CV than in the GF condition (see data in next section). However, the percentage of TCR{alpha}ß+ LPLs that expressed CD4 (median, 54%) was significantly smaller in GF than in CV animals (P < 0.01). The same was true (P < 0.01) for the percentage of the TCR{alpha}ß+ IELs that expressed CD4 in GF (49%) versus CV rats (Figure 1). Conversely, the proportion of TCR{alpha}ß+ LPLs that expressed CD8 (38%) in GF rats was significantly higher than that in CV rats (P < 0.001). The fraction of TCR{alpha}ß+ IELs expressing CD8 (42%) was likewise significantly higher (P = 0.016) in GF than in CV animals (Figure 2).

The estimated area of NALT at corresponding section levels was only marginally, although significantly (P < 0.01), smaller in GF than in CV rats (Figure 3), and, in both cases, the lymphoid aggregates consisted of T cells (mainly CD4+) and primary B-cell follicles without recognizable germinal centers (Figures 4A and 4C). This observation agrees with the recent report that the normal microflora of the nose provide insufficient immunostimulation to induce germinal centers also in murine NALT (17), whereas the gut flora drives germinal center formation in rat Peyer's patches (10).



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  		Figure 3. The section area size of organized NALT before and after monoinfection with M. pulmonis in germ-free and conventional rats. Medians and significant differences are indicated. Open circles, uninfected; closed circles, infected, 3 wk.

 


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  		Figure 4. Multicolor immunofluorescence localization of T cell subsets and B cells in the nose of GF control rats and GF rats monoinfected with M. pulmonis for 3 wk. Tissue sections were immunostained for TCR{alpha}ß, CD3, CD4, CD8, NKR-P1 (NK-cell marker), B cells, and epithelium (cytokeratin, CK) in different combinations (see color keys). (A and B) In the controls, NALT aggregates are much less prominent than after monoinfection for 3 wk, when the dominance of CD4+ T cells with purely red color becomes even more apparent than before. Note that in both situations there are several purely green CD8+ cells (mostly with NK phenotype; see E), and these also occur in the epithelium after infection, together with purely red (CD4+) intraepithelial T cells (arrows). The red apical staining in the epithelium is nonspecific. (C and D) The hypertrophy of the NALT aggregates is largely caused by follicular hyperplasia of B cells with expanded germinal centers (GCs) and mantle zones (MZs), whereas, in the control state, the follicles are mainly of primary type without GCs. Note that the B-cell marker detected by mAb OX33 is downregulated on GC B cells. The T cells show an abundant blue-green color mix, verifying their preferential CD4 phenotype, particularly inside of GCs. The few purely green subepithelial cells (arrows in D) represent CD4+ macrophages or dendritic cells. (E) Many of the CD8+ cells, both in the lamina propria and surface epithelium, express the NKR-P1 marker, and therefore appear reddish or yellow, but there are also purely red NKR-P1+ cells without CD8 expression. Original magnification: A–D, x100; E, x200.

 
Mycoplasma pulmonis Infection Induces Expansion of NALT and Mucosal T Cells
The cross-sectional dimension of NALT increased ~ 2.6-fold in CV rats under the influence of mycoplasma infection (P < 0.01), and such NALT hypertrophy tended to be relatively more marked (5.2-fold; P < 0.01) in GF rats (Figures 3 and 4B). Thus, in the postinfection state, there was no difference between CV and GF animals in the calculated section area of NALT. Immunostaining in both CV and GF animals showed that, compared with the uninfected state (Figures 4A and 4C), the mycoplasma-induced NALT hypertrophy included aggregated hyperplastic B-cell follicles with germinal centers rich in CD4+ T cells (Figures 4B and 4D), which were easily distinguishable from the diffusely distributed B and T lymphocytes seen elsewhere in nasal mucosa.

A significant numerical elevation of TCR{alpha}ß+ LPLs per millimeter surface epithelium was seen after mycoplasma infection in nasal mucosa of both CV (7.2–10.8; P = 0.008) and GF (7.1–12.9; P = 0.008) rats, and the same was true for TCR{alpha}ß+ IELs in GF rats (1.0–1.7; P = 0.02), but not in CV rats (1.7–1.8; P = 0.31). In the postinfection state, the estimated number of TCR{alpha}ß+ LPLs and IELs did not differ between the two animal groups. The rare TCR{gamma}{delta}+ T cells were apparently not numerically or phenotypically affected by the infection. These results harmonized with the T-cell distribution observed in rat laryngeal mucosa of CV rats after inhalation of heat-killed Moraxella catarrhalis (26).

M. Pulmonis Infection Alters Nasal T-Cell Phenotypes in Germ-Free but Not in Conventional Rats
Monoinfection with M. pulmonis induced a striking proportional increase of TCR{alpha}ß+CD4+ T cells in GF rats, both in the lamina propria (P < 0.01) and in the epithelium (P < 0.01), whereas this LPL and IEL subset ratio did not change in CV rats (Figure 1). Conversely, the percentage of TCR{alpha}ß+CD8+ in both compartments was significantly decreased in GF (P < 0.01) but remained virtually unchanged in CV rats (Figure 2). Notably, in contrast to those in the pre-infection state, the infected rats showed a significantly higher proportion of the TCR{alpha}ß+ LPLs expressing CD4 in the GF than in the CV condition (P = 0.03). The same striking difference between the two conditions was observed for TCR{alpha}ß+ IELs expressing CD4 when comparing the pre-infection with the post-infection state (P < 0.01) (Figure 1).

A Subset of Nasal Lymphocytes Expresses the NK-Cell Complex
Interestingly, we found a relatively large population of nasal CD8+ lymphocytes with no detectable TCR{alpha}ß (Figure 4B) or TCR{gamma}{delta} (data not shown). The fraction of TCR{alpha}ß–CD8+ of all CD8+ IELs was 53% in GF rats and 62% in CV rats, whereas in the lamina propria these fractions were 57% and 74%, respectively. Many of these cells coexpressed the NK-cell marker NKR-P1 (Figure 4E) and were also CD2+; they could thus be phenotypically characterized as NK cells. In CV rats, this subset tended to be decreased after M. pulmonis infection, whereas no difference was observed in GF animals (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This is apparently the first study to show a local immune-modulating effect of commensal bacteria in the face of infection with an extracellular respiratory pathogen. Over time, M. pulmonalis is known to cause chronic inflammatory disease in the airways of rodents. Although specific T-cell responses could not be evaluated in this in situ study, we found that the monoinfection induced more severe perturbations of immunologic variables in GF than in CV rats (e.g., by affecting both the number and proportion of CD4+ TCR{alpha}ß cells in the lamina propria and surface epithelium). Thus, mucosal immune homeostasis was better maintained during the infection in the presence of the indigenous microbiota, and this was also reflected by less hypertrophy, in relative terms, of the local inductive NALT structures in CV than in GF rats.

The host's coexistence with commensal bacteria in a mutually beneficial manner has developed over several million years of adaptation (7). The immunologic hyporesponsiveness to the indigenous flora is believed to reflect a tolerance phenomenon (27), and may largely depend on regulatory T (Treg) cells secreting transforming growth factor–ß and interleukin-10, which may suppress immune responses. These cytokines inhibit both T helper types 1- and 2-dependent immunity by dampening both T-cell–mediated as well as T-cell–independent immunopathology (28, 29). There is currently great interest in the role of APCs in shaping the phenotypes of naive T cells during their initial priming because differential expression of costimulatory molecules on both steady-state and activated dendritic cells (DCs) can exert a decisive impact. Thus, the function of DCs is modulated by pathogen-associated molecular patterns, which are sensed by pattern recognition receptors (PRRs)—many of which belong to the so-called Toll-like receptors. The engagement of PRRs on DCs causes maturation accompanied by production of cytokines and upregulation or downregulation of cell-surface molecules according to strictly defined kinetics (30). Such signaling molecules will critically influence further induction of both innate and adaptive immunity.

By means of their pathogen-associated molecular patterns, pathogens can quite early during an infection imprint their "signatures" on subsequent immune responses. Treg cells may even be directly affected by microbial products, such as lipopolysaccharide, through the Toll-like receptors that they express (31). It is important to be aware of the fact that PRRs apparently do not distinguish between pathogenic and commensal bacteria, which might seem incompatible with the mucosal homeostasis that normally exists. However, it cannot be excluded that the indigenous flora may induce differential PRR signals, resulting in distinct molecular APC programs (32). Notably, it has been reported that nonpathogenic Salmonella strains are able to block the nuclear factor–{kappa}B transcription pathway in human gut epithelial cells in vitro and thereby reduce basolateral interleukin-8 secretion in response to proinflammatory stimuli, including apical infection with wild-type S. typhimurium (13). It is also notable in this context that the intestinal epithelium appears to have inherent mechanisms to protect itself against activation from the luminal side unless production of chemokines and proinflammatory cytokines is needed in defense against invading microorganisms (33). Thus, mucosal epithelial cells apparently possess sensing systems that allow discrimination between pathogenic and nonpathogenic bacteria to initiate an inflammatory reaction only when elimination of invading pathogens is needed. The secretory immune system appears to be part of this homeostatic mechanism because specific dimeric IgA may, during polymeric Ig receptor–mediated epithelial transcytosis to the mucosal surface, prevent lipopolysaccharide-induced nuclear factor–{kappa}B translocation and induction of proinflammatory cytokines (34).

Further work is needed to investigate whether the dampening effect of the normal microbiota on pathogen-induced immunologic perturbations observed in our study was mediated by Treg cells or other homeostatic mechanisms alluded to above. Notably, it was recently shown in a rat model that the response to inhaled allergens in sensitized animals is regulated via bidirectional interactions between antigen-presenting DCs and memory T cells (35). After aeroallergen challenge, the resident DCs rapidly matured in situ to potent APCs. This model provided a plausible model for a regulatory role of activated CD4+ T cells in airway mucosa.

The commensal flora in the human nasopharynx is similar to the better-known microbiota of the mouth and oropharynx. The numbers and prevalence of various bacteria in the nasopharynx, including different species of Streptococcus (i.e., S. mitis, S. pneumoniae, S. oralis, and S. viridans) vary individually and appear to depend on age (36). During different periods of life, colonization with potential pathogens is known in the nasopharynx. In childhood, S. pneumoniae, Hemophilus influenzae, and Moraxella catharalis are relatively frequent in the nasopharyngeal region and elevated in populations with, for instance, otitis media. Approximately 30% of adults harbor Staphylococcus aureus in the most anterior region of the nasal cavities. Bachert and colleagues (37) have suggested that an aberrant immune response of exotoxin from S. aureus results in the formation of local IgE antibodies in association with nasal polyps. Increased numbers of S. aureus and other transient pathogens (i.e., Pseudomonas spp.) in the sinonasal region and lower respiratory tract, in the case of cystic fibrosis, could be associated with an increased prevalence of allergic eosinophilia in this population (38).

Whether the aberrant response patterns referred to above are explained by inappropriate immunoregulatory signals from the indigenous microbiota remains elusive. According to a recent Danish study, the normal flora of the human nasal cavity consists of Corynebacterium, Aureobacterium, Rhodococcus, and staphylcocci, including Staphylcoccus epidermidis, S. capitis, S. hominis, S. haemolyticus, S. lugdunensis, and S. warneri (39). This composition differs totally from the nasopharyngeal flora. Notably, the status and role of the commensal flora in the human nose and nasopharynx remain unknown in chronic or recurrent infection and inflammation.

In the case of rodents, the colonization of microbes in the nasal cavity and nasopharyngeal region appears to be more prominent than in humans, and rats breathe mainly through the nose. This might explain that we achieved such a striking effect on pathogen-induced immunologic perturbations when comparing GF with CV rats. The fact that the nose and nasopharynx are quite exposed to the external environment, being in persistent contact with irritants and antigens, makes it most likely that the immunologic impact of the microflora in this region represents an important homeostatic mechanism similar to that known to exist in the gut (5–7). Thus, in our rat model, we demonstrated that the normal microbiota modulates the response of various T-cell subsets (i.e., TCR{alpha}ß+ CD4+ and CD8+ cells) during monoinfection with the pathogen M. pulmonis. This model may be further exploited to dissect immunoregulatory network components engaged by commensal bacteria in the airways.


   Acknowledgments
 
This work was supported by the Swedish Medical Research Council (project 00749), the Karolinska Institute Research Foundation, the Vårdal Foundation, the Swedish Heart Lung Foundation, the Norwegian Cancer Society, the Research Council of Norway, the University of Oslo and Anders Jahre's Fund. We thank the technical staff at the Laboratory of Microbial Ecology, Karolinska Institute, Stockholm, and the technical staff at LIIPAT, Institute of Pathology, Rikshospitalet University Hospital, Oslo. Hege Eliassen and Erik K. Hagen are acknowledged for expert secretarial assistance with the manuscript.


   Footnotes
 
Conflict of Interest Statement: G.H. has no declared conflicts of interest; L.H. has no declared conflicts of interest; T.M. has no declared conflicts of interest; P.S. has no declared conflicts of interest; and P.B. has no declared conflicts of interest.

Received in original form August 25, 2004

Received in final form September 13, 2004


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Brandtzaeg, P. 1995. Immunocompetent cells of the upper airway: functions in normal and diseased mucosa. Eur. Arch. Otorhinolaryngol. 252:S8–S21.
  2. Fokkens, W. J., A. F. Holm, E. Rijntjes, P. G. Mulder, and T. M. Vroom. 1990. Characterization and quantification of cellular infiltrates in nasal mucosa of patients with grass pollen allergy, non-allergic patients with nasal polyps and controls. Int. Arch. Allergy Appl. Immunol. 93:66–72.[Medline]
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