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(Journal of Leukocyte Biology. 2001;69:381-386.)
© 2001 by Society for Leukocyte Biology

MHCII, Tlr4 and Nramp1 genes control host pulmonary resistance against the opportunistic bacterium Pasteurella pneumotropica

Stephen K. Chapes^*, Derek A. Mosier, Albion D. Wright^* and Marcia L. Hart^*

^* Division of Biology
Department of Diagnostic Medicine and Pathobiology, Kansas State University, Manhattan

Correspondence: Stephen K. Chapes, Ph.D., Kansas State University, Division of Biology, Ackert 19, Manhattan, KS 66506-4901. E-mail: skcbiol{at}ksu.edu

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
MHCII, Tlr4, and Nramp1 genes are each independently important in pulmonary immunity. To determine the effect of these genes on host resistance, mice carrying various combinations of functional alleles for these three genes were experimentally challenged with the opportunistic bacterium, Pasteurella pneumotropica. MHCII^-/-, Tlr4^d/d, and Nramp1^s/s mice were significantly more susceptible to experimental infections by P. pneumotropica after intranasal challenge compared to mice carrying functional alleles at only one of those genes. P. pneumotropica were cultured from the lungs of challenged mice, and the severity of the pneumonia strongly correlated with the number of isolated bacteria. Mice with the genotype MHCII^-/- Tlr4^n/n genotype were less susceptible to pneumonia than MHCII^+/+, Tlr4^d/d mice. It is interesting that the Nramp1 gene contribution to host resistance was apparent only in the absence of functional MHCII or Tlr4 genes. These data suggest that MHCII, Tlr4, and Nramp1 genes are important to pulmonary bacterial resistance.

Key Words: genotypes • lungs • mice

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Many genes contribute to a functional immune system [¹ ]. Studies with knockout and transgenic mice are commonly used to investigate host responses when specific genes are absent or over-expressed. However, few models address the more complex issue of multigenic control of host defense. During the creation of mice without functional genes at MHCII and Lps (subsequently defined as the toll-like receptor 4, Tlr4 [² , ³ ]) loci, we discovered one of the newly created strains had a high incidence of fatal pneumonia associated with Pasteurella pneumotropica infection. P. pneumotropica is a ubiquitous, gram-negative bacterium that is present in most rodent colonies and is of minimal significance in immunocompetent mice. However, we found that mice lacking functional alleles at MHCII, Tlr4, and Nramp1 genes were highly susceptible to spontaneous P. pneumotropica-induced pneumonia compared to mice that have functional forms of at least one of these three genes.

The purpose of this study was to test the hypothesis that mice lacking functional alleles at MHCII, Tlr4, and Nramp1 genes would be susceptible to experimental P. pneumotropica-induced pneumonia. We report the variable susceptibility of mice with different combinations of MHCII, Tlr4, and Nramp1 to experimental challenge with P. pneumotropica. We also assess the relative importance of these genes in resistance to P. pneumotropica pneumonia.

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Mice
Names and genotypes of the mice reported in this study are listed in Table 2. All of the mice were bred in the rodent facility of the Division of Biology at Kansas State University. C3HeB/FeJ (FeJ), C3H/HeJ (HeJ), and C57BL/6J (B6) mice were originally obtained from Jackson Labs (Bar Harbor, ME). C2D mice, originally described in 1991, are on the C57BL/6 background [⁴ ]. These mice have maintained the MHCII^-/- genotype through 19 consecutive brother-sister matings at Kansas State University. The C2D mice served as founders for all five of the recombinant mouse strains described in this study. C57BL10/ScN (BL10) mice were obtained from the animal resource facility at the NIH and served as founders for the B10 x C2D recombinant stock. HeJ mice and FeJ mice served as founders for HeJ x C2D and FeJ x C2D, respectively. The creation of B10 x C2D, HeJ x C2D(S), and HeJ x C2D(R) mice has been described previously [² ]. The FeJ x C2D(S) and FeJ x C2D(R) mouse strains were created to serve as MHCII^-/-, Tlr4^n/n, Nramp1^s/s, or MHCII^-/-, Tlr4^n/n, Nramp1^r/r comparison to the HeJ x C2D(R) and HeJ x C2D(S) mouse strains. These mice were generated by crossing C2D with C3HeB/FeJ mice using techniques described previously [² ]. The B10 x C2D mice are essentially syngeneic with only three known allelic differences between C57BL/6 and C57BL/10 mice [⁵ ]. The other recombinant mice have been maintained by brother-sister matings but with the exception of MHCII, Tlr4, and Nramp1 their genotypes are mixed because these mice have not been backcrossed into the parental backgrounds. All recombinant strains were housed in the mouse facility in an isolator under specific pathogen-free conditions. Recombinant breeder mice are given sulfamethoxazole and trimethoprim (Sulfatrim^®, 1 mL/100 mL of H₂O) for a period of 1 week once per month to inhibit infections. Weaned mice do not receive antibiotics. All mouse experiments were approved by the institutional animal care and use committee.

Infections
P. pneumotropica A1 (American Type Culture Collection no. 35149) was grown on brain-heart infusion agar plates with 5% sheep blood, and colonies were selected and transferred to brain-heart infusion broth for overnight growth on a rotary shaker (180 rpm at 37°C). After transfer to fresh medium and growth for an additional 6–8 h, cultures were centrifuged at 10,000 rpm for 15 min and resuspended in sterile phosphate-buffered saline (PBS). Mice were intranasally infected [⁶ ] by inducing gasping with exposure to a 50/50 carbon dioxide (CO₂) to oxygen (O₂) mixture, application of 25 µL of culture, to the nostril (4 x 10⁸ to 3 x 10¹¹ CFU), followed by an additional 10-s exposure to the CO₂/O₂ mixture.

Lung assessment
One to three mice were killed by cervical dislocation at the times indicated for each experiment. Immediately after death, lungs were removed, placed in individual tubes, and iced. The percentage of pneumonic tissue for each lung lobe was determined with the use of a dissection microscope and multiplied by 2 (left lung lobe), 1 (right superior and right inferior lobes), or 0.5 (right middle and post caval lobe) to account for the relative contribution of each lobe to the total pulmonary mass. The individual lesion score is the sum of the scores for all lung lobes in a mouse. The total lesion score is the sum of the individual lung scores of all the mice in each strain. After evaluation, lung samples were collected for bacterial culture and polymerase chain reaction (PCR). Bacteria were plated onto blood-agar plates and observed for growth and organism identification after 24-h incubation at 37°C. Other lung samples were homogenized as previously described [⁷ , ⁸ ] and 10-µL samples were subjected to PCR amplification using the P. pneumotropica 16S ribosomal RNA gene-specific primers: 5’-CGGAATAACTGGGCGTAA-3’ (upstream); 5’-GTCTCCTTTGAGTTCCCGACC-3’ (downstream, National Center for Biotechnology Accession no. AF012090). The mammalian housekeeping gene S14 [⁹ ] was amplified with an identical sample of lung DNA as a control to estimate the amount of host tissue present. Tissue from some lungs was fixed in 10% neutral buffered formalin for histopathological examination. After fixation, lungs were processed routinely, embedded in paraffin, 5- to 6-µm sections were prepared, and staining was with hematoxylin and eosin.

Alveolar macrophages-nitrite assessment
To assess alveolar macrophage function we estimated the production of nitric oxide by the detection of the stable, oxidized nitrite end product. Two mice per group were intranasally infected with 5 x 10⁶ CFU of bacteria as described above. Four days later, mice were killed with CO₂ and their tracheas were exposed. Each trachea was cannulated with a blunt-end needle and tied in place using surgical thread. Lungs were filled with 1.0 mL of PBS/EDTA (0.2 g/L, 25°C) using a 1-mL syringe and gently massaged. Lavage fluid was aspirated and placed on ice in 15-mL polypropylene centrifuge tubes. Macrophages (5 x 10⁵) were seeded in wells of 24-well plates and incubated for 2 h at 37°C. Wells were gently washed with warm RPMI containing 5% fetal bovine serum (FBS) to remove nonadherent cells. Remaining cells were incubated for 18 h. Clarified supernatants were assessed for the presence of nitrite with the use of the Greiss reagent [¹⁰ ].

Statistics
Differences between treatment groups were determined using the indicated test analyses in the StatMost Statistical Package (DataMost, Salt Lake City, UT).

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Spontaneous infections in recombinant strains
Recombinant MHCII^-/-, Tlr4^d/d mice were originally developed to examine the relationship between MHCII and Lps (Tlr4) loci in superantigen-mediated macrophage activation studies [³ ]. Spontaneous pneumonia caused by opportunistic P. pneumotropica was recognized in the recombinant mice soon after their development. Mortality in these mice was particularly common in mice on the "BL" background (Table 1 ). Because Nramp1 plays an important role in macrophage function [¹¹ ], this observation led to selection for homozygous expression of Nramp1^r or Nramp1^s alleles in HeJ x C2D mice [² ] and the creation of recombinant mice for experimental challenge studies.

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Table 1. Death Rates and Age of Onset by Spontaneous Pneumonia BL10xC2D and HeJxC2D Mice

Experimental challenge with P. pneumotropica
To directly assess the role of Nramp1 along with MHCII and Tlr4 genes in the control of pneumonia, experimental infections with P. pneumotropica were conducted on several mouse strains, including HeJ x C2D mice selected for the homozygous expression of Nramp1^s/s and Nramp1^r/r genotypes [² ]. Post-challenge recovery of bacteria from the lungs after infection with 4 x 10¹⁰ CFU are shown for bacteriologic cultures (Table 2 ) and PCR of the P. pneumotropica-specific S16 ribosomal gene (Fig. 1 ). Bacterial burdens were highest in HeJ x C2D(S), HeJ, B10, and B10 x C2D mice and bacteria were recoverable from all B10 x C2D mice. The severity of pneumonia based on lesion scores was predictable according to each mouse genotype (Table 2) . Grossly, lesions consisted of areas of bronchopneumonia, which progressed to large, multifocal to coalescing abscesses. Figure 2A demonstrates unaffected lung. In affected lungs, pneumonia originated within small airways and adjacent alveoli (Fig. 2B) , progressing to regional bronchopneumonia that affected all airways and alveoli (Fig. 2C) . Subsequently, normal pulmonary architecture was obliterated by the development of large, coalescing abscesses (Fig. 2D) . One hundred percent of B10 x C2D mice developed pneumonia, 3 of 14 of which died during the experiment. For HeJ x C2D(S) mice, 54% developed pneumonia and 1 of 13 mice died during the experiment. Although 54% of the C3H/HeJ mice and 43% of the C57Bl/10ScN mice (Tlr4^d/d) developed pneumonia, none of these mice died and the progression and severity of pneumonia decreased with time (Table 3 ). No mice of any other strain died at this challenge dose. There was a 7% incidence of pneumonia in HeJ x C2D(R) mice with no fatalities (Table 2) . For all mouse strains, there was a strong correlation between the number of P. pneumotropica that could be cultured from lung samples and the severity of the pneumonia (r = 0.783). Moreover, when alveolar macrophages were assessed for their ability to produce nitric oxide after a sublethal P. pneumotropica challenge, there was a correlation between the Tlr4 genotype and the ability to make nitric oxide after infection (Table 4 ).

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Table 2. Severity of Infection in Affected Mice after Experimental Challenge with P. pneumotropica: Pathology and Bacteriology

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Figure 1. PCR for P. pneumotropica DNA (lanes marked P) for selected mouse samples 26 days after infection with 4 x 1010 CFU bacteria. Loading controls PCR for the S14 ribosomal housekeeping gene (lanes marked S).

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Figure 2. (A) Uninfected lung, stained with hematoxylin and eosin. An example of two terminal bronchioles opening into alveolar ducts. Bronchiolar and alveolar lumens are devoid of inflammatory cells; original magnification x80. (B) Early pneumonia in an MHCII-/-, Tlr4d/d, Nramp1s/s mouse. Terminal bronchioles and adjacent alveoli that contain large numbers of neutrophils. Section stained with hematoxylin and eosin; original magnification x120. (C) Advanced pneumonia in an MHCII-/-, Tlr4d/d, Nramp1s/s mouse. Pneumonia is characterized by large numbers of neutrophils filling the lumens of bronchi, bronchioles, and alveoli. Section stained with hematoxylin and eosin; original magnification x40. (D) Chronic pneumonia in an MHCII-/-, Tlr4d/d, Nramp1s/s mouse. This advanced stage of pneumonia is characterized by focal abscesses that obliterate the normal pulmonary parenchyma. Abscesses consist of central cores of cellular debris and degenerate neutrophils surrounded by a fibrous capsule. Section stained with hematoxylin and eosin; original magnification x60.

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Table 3. Pathological Assessment of Mice Infected with P. pneumotropica

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Table 4. Production of Nitric Oxide after P. pneumotropica Administration

To confirm that C2D mice can resolve P. pneumotropica infections better than B10 mice, we challenged C2D, B10, B6, and B10 x C2D mice with 4 x 10⁸ bacteria. These mice are essentially syngeneic [⁵ ] and the impact of the infection on the development of pneumonia could be focused on differences in MHCII and Tlr4 genes. Bacterial DNA was detectable in B6, B10, and B10 x C2D mouse lungs by PCR as late as 10 days, whereas C2D mice cleared bacteria by 10 days. Lesions in B6 mice were minimal, subtle, and only present on day 6; in C2D mice there were small abscesses present on days 6 and 7 (Table 5 ). In contrast, there were coalescing abscesses on a large percentage of the lung lobes of B10 and B10 x C2D mice on post-challenge days except day 1 (Table 5) .

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Table 5. Lesion Assessment of Mice Infected with P. pneumotropica

To determine whether functional genes at MHCII, Tlr4, or Nramp1 would be protective at higher bacterial challenge doses, mice carrying several allelic combinations of MHCII, Tlr4, and Nramp1 were challenged with 10¹¹ CFUs of P. pneumotropica. Mice with any combination of one or two functional MHCII, Nramp1, or Tlr4 genes were significantly more resistant to fatal pneumonia compared to mice that were deficient in all three genes. By 20 days post-challenge with 3 x 10¹¹ CFU, 8 of 10 HeJ x C2D(S) mice died compared to 1 of 10 HeJ x C2D(R) mice at this challenge dose. No deaths occurred in the HeJ, FeJ, FeJ x C2D(R), or FeJ x C2D(S) groups (Fig. 3 ). Similarly, when B10, B6, C2D, and B10 x C2D mice were challenged with 1 x 10¹¹ CFU of P. pneumotropica, 4 of 5 B10 x C2D mice were dead by 10 days post-challenge and all other mice survived for the duration of the 10-day experiment (Fig. 4 ).

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Figure 3. Survival of mice having various allelic combinations of MHCII, Tlr4, and Nramp1after infection with P. pneumotropica. Mice were infected intranasally with 3 x 1011 P. pneumotropica. Mice with any combination of single or double mutations at MHCII, Nramp1, or Tlr4 were significantly more resistant to Pasteurella-induced pneumonia. Recombinant mice challenged with 3 x 1011 P. pneumotropica intranasally. FeJ x C2D mice are MHCII-/-, Tlr4n/nm, Nramp1r/r, or Nramp1s/s. n = 10 mice per group.

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Figure 4. Survival of Nramp1s/s mice having various allelic combinations of MHCII and Tlr4 after infection with 1 x 1011 CFU of P. pneumotropica. Recombinant mice challenged with P. pneumotropica intranasally. n = 5 mice per group.

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In immunocompetent mice, P. pneumotropica is normally a nonpathogenic, commensal bacterium. However, in mice with one or more nonfunctional alleles at MHCII, Tlr4, and Nramp1, spontaneous and experimental P. pneumotropica infection results in pneumonia and sometimes death. The nature and pathogenesis of the pneumonia produced by the challenge was typical of naturally occurring bronchopnuemonia. In some mice, the pneumonia developed slowly and did not progress. In mice with deficiencies in all three genes, the pneumonia progressed rapidly, ending in relatively acute death due to fulminate bronchopneumonia, or in mice killed in later stages of the study, more mature pneumonia characterized by abscess formation that may or may not have been fatal. The need to disrupt the immunophysiological components that are controlled by three distinct genes to induce severe P. pneumotropica pneumonia exemplifies the immunological redundancy of the lung.

Our observations are consistent with the independent roles that MHCII and Tlr4 are thought to play in lung immunity. In AIDS patients, the loss of MHCII-controlled CD4⁺ T cells is associated with the onset of opportunistic lung infections [¹² ]. Tlr4^d/d mice are more susceptible to gram-negative, mucosal infections than Tlr4^n/n mice [¹³ ]. Our findings are also consistent with the regulatory role Tlr4 has on macrophages [¹⁴ ] and the role that macrophages play in lung immunity. When alveolar macrophages were ablated in normally resistant C57BL mice [¹⁵ ] these mice became susceptible to mycoplasma infections [¹⁶ ]. These genes can either directly influence macrophage activation (Tlr4; [^{17 18 19} ]), cytokine stability, or mediator production (Nramp1; [¹¹ , ²⁰ , ²¹ ]) or CD4⁺ T cells that produce cytokines such as interferon- that are necessary for adequate macrophage activation [⁴ , ^{22 23 24} ]. Therefore, it is not surprising that dysfunctional combinations of genes that are important for macrophage and T cell function (MHCII, Nramp1 and Tlr4) lead to increased susceptibility to opportunistic lung infections.

The resistance of mice carrying functional alleles in at least one gene locus of MHCII, Tlr4, or Nramp1 suggests that the macrophage and T cell functions controlled by these genes are independent, but can compensate for each other. Moreover, the data suggest a hierarchy of these three genes in host defense against P. pneumotropica infection. Because Nramp1^s/s mice do not develop Pasteurella-induced pneumonia, when either MHCII genes or the Tlr4 genes are functional, we consider it to be the least important gene in the hierarchy, which would be consistent with previous, controversial observations on the role of Nramp1 in lung immunity [²⁵ , ²⁶ ]. However, there is a more complex relationship between MHCII and Tlr4. When Tlr4 is nonfunctional, even in the presence of MHCII, B10 and HeJ mice develop experimental pneumonia. When MHCII was nonfunctional and Tlr4 was normal (i.e., C2D mice or FeJ x C2D mice), mice rarely developed pneumonia. These data suggest that Tlr4 is more important to the host response than MHCII. However, even though Tlr4^d/d, MHCII^+/+ mice develop pneumonia, the pneumonia does not progress, and it is not lethal. Lethal pneumonia occurred only when both MHCII and Tlr4 were nonfunctional. Therefore, functional CD4⁺ T cells seem to compensate for the reduced macrophage response in Tlr4^d/d mice, suggesting a more important role for MHCII in the host response against P. pneumotropica. Because of the interdependence of MHCII and Tlr4, an appropriate ranking of the gene importance is: (MHCII = Tlr4) > Nramp1. These data also indicate that the innate immune responses controlled by the Tlr4 gene are very important to the successful resolution of lung infections by some opportunistic pathogens.

The availability of mice that carry functional and nonfunctional genes at MHCII, Tlr4, and Nramp1, and that are highly susceptible to P. pneumotropica infection provides an ideal model for studying and understanding host resistance to opportunistic respiratory infections. The precise reasons for the increased susceptibility of MHCII^-/-, Tlr4^d/d, Nramp1^s/s mice to P. pneumotropica challenge have not been completely characterized. The loss of CD4⁺ T cells in MHCII^-/- mice fails to provide a sole explanation for the increased susceptibility to pneumonia. Perhaps in the MHCII^-/- mice, CD8⁺ T cells are able to compensate for the absence of helper T cells as has been seen in Salmonella infections [²⁷ ]. However, indications from the measurements of NO production by alveolar macrophages from Tlr4^d/d mice after P. pneumotropica challenge (Table 4) suggest that pulmonary macrophage function is compromised because of defects in the Tlr4 gene-associated signaling cascade. The inability of Tlr4^d/d mice to respond to the bacterial lipopolysaccharide may put them at a significant disadvantage early in the infection. When CD4^+/+ T cells are present they can overcome that problem but in their absence the bacteria go unchecked and the animals develop severe-to-lethal pneumonia. Future experiments will more precisely characterize the various immunological components that contribute to the increased susceptibility of the mice to opportunistic pulmonary pathogens.

 
This work was supported by the American Heart Association Grant KS-97-GS-02, NASA grants NAGW-1197, NAGW-2338, NAG2-1274, and NCC5-168, United States Department of Agriculture Animal Health Funds 1433 Grant 4-81895, NIH Grant CA09418, and the Kansas Agricultural Experiment Station. This is Kansas Agriculture Experiment Station publication number 00-87-J. The authors thank Dr. Allison Beharka, Dr. Dorothy Feese, Dr. Allan Forsman, Ms. Angie Herpich-Matthews, and Ms. Donna Rogers for their assistance in various stages of this work.

Received August 20, 2000; revised November 8, 2000; accepted November 9, 2000.

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