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Immunology Unit, Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom
Tumor necrosis factor (TNF) has been proposed as a major mediator of host resistance in murine models of Streptococcus pneumoniae infection; in humans, anti-TNF therapies have been implicated in increased susceptibility to pneumococcal infection. Here, we use nonlethal (serotype 6B) and lethal (serotype 3) S. pneumoniae, neutralizing monoclonal antibodies to TNF, and TNF genedeficient mice to reexamine the role played by TNF in antistreptococcal responses. After nonlethal challenge, primary resistance and all examined parameters of the cellular inflammatory response occurred independently of TNF activity. After lethal challenge, TNF deficiency resulted in more-rapid death but did not affect lung inflammation. However, the livers of the TNF genedeficient mice, but not of the control mice, exhibited extensive signs of systemic disease. TNF, therefore, is dispensable for a complete cellular pulmonary inflammatory response to S. pneumoniae infection but enhances survival from disseminated lethal infection, at least in part by delaying systemic organ damage.
Pneumococcal diseases caused by the gram-positive bacterium Streptococcus pneumoniae, including pneumococcal pneumonia, septicemia, and meningitis, result in >1.2 million deaths each year worldwide [1]. With dual antibiotic resistance among pneumococci in the United States predicted to reach 41% by July 2004 [2] and with increased susceptibility among the young, the elderly, those with underlying chronic diseases, and those undergoing elective immunosuppression, there remains a pressing need to understand pneumococcal disease pathogenesis [3].
Despite the clinical importance of S. pneumoniae, host responses to this organism are relatively poorly understood. In mice, host resistance to S. pneumoniae has been most commonly studied after infection with highly encapsulated serotypes, such as 2 and 3. In most common mouse strains, infection with these serotypes leads to almost uniform fatality [4]. In this setting, it has been shown that primary responses to S. pneumoniae infection involve cytokines, including tumor necrosis factor (TNF) [57], interleukin (IL)1 [6], IL-18 [8], and IL-6 [9]. Of these, TNF is widely reported to be a critical component in host defence against intranasal (inl) pneumococcal infection. Compared with control mice, TNF receptor (TNFR) type I genedeficient mice [5] and mice given anti-TNF antibodies [57] exhibit elevated bacteremia and progress more rapidly to a fatal outcome. Similarly increased susceptibility also results from TNF deficiency during peritoneal, bacteremic, and intracranial S. pneumoniae infection [1012]. However, the cause of death in these studies was not determined, and the role played by TNF in the immune response to S. pneumoniae infection remains undefined.
At the cellular level, protection likely results from a balance of bacterial clearance by host phagocytes and bacterial escape through replication and capsule production [13]. The early immune response to S. pneumoniae infection is phagocyte dominated [1315], although the relative contributions of neutrophils and macrophages are undetermined. Despite putative roles for NK T cells in regulating early phagocyte recruitment and TNF production [16], neither the pulmonary source of TNF nor the role played by TNF in regulating host-protective cellular events during S. pneumoniae infection have been resolved.
The present study examines the role of TNF by use of inl infection with S. pneumoniae serotypes 6B and 3, strains that result in lung-restricted, nonlethal infection and disseminated, lethal infection, respectively. We demonstrate that, during nonlethal infection, pulmonary macrophages are the major source of TNF. Strikingly, we found no evidence for a role of TNF in either regulation of inflammatory responses or bacterial clearance during nonlethal infection. After lethal S. pneumoniae challenge, TNF deficiency did not significantly affect pulmonary inflammation but resulted in a clear exacerbation of hepatic pathological features that was associated with more-rapid progression to death. TNF may, therefore, be dispensable in pulmonary antistreptococcal responses but a necessary beneficial regulator of systemic organ protection in this disease.
MATERIALS AND METHODS
Mice and bacteria.
Male DBA/1 mice were supplied by Harlan UK. C57/BL6 (B6) and B6 TNF genedeficient (TNF-/-) mice were bred under barrier conditions at the London School of Hygiene and Tropical Medicine (LSHTM). Mice, supplied with food and water ad libitum, were used at 812 weeks of age and were killed when a fatal outcome was deemed to be inevitable. Animal experimentation was performed with approval from the LSHTM Animal Procedures Ethics Committee and the United Kingdom Home Office.
Clinical isolates of S. pneumoniae serotypes 6B and 3 (Microbiology Department, Royal Free Hospital, London, United Kingdom) were cultured overnight from frozen glycerol stocks in brain-heart infusion broth (Oxoid) in a CO2-enriched atmosphere (Campygen; Oxoid) and then were washed and resuspended in sterile PBS. Infective doses were determined from colony counts after serial-dilution plating of the inoculum on Colombia blood agar plates (Oxoid). Mice were inl immunized with 50 L of inoculum under light anesthesia, as described elsewhere [14].
TNF blockade.
TNF was neutralized by use of either the monospecific antimurine TNF- monoclonal antibody (MAb) cV1q [17] (Centocor) or the murine TNFR immunoglobulin (TNFR-Ig) fusion protein p75P-sf3, which is analogous to the previously described human p75-sf3 [18], with the human p75 and IgG1 domains replaced with the murine p75 and IgG2a sequences, respectively (Centocor). Control mice received isotype control antibody (cVam; Centocor). Mice were administered 0.5 mg of MAb or fusion protein intraperitoneally (ip) 11 and 4 days before challenge and, when necessary, 3 days after challenge. The in vivo efficacy of both blocking reagents was confirmed by use of a model of collagen-induced arthritis [19]. Treatment 14 days after the onset of arthritis with either cV1q or TNFR-Ig, but not with cVam, blocked subsequent disease progression and significantly reduced clinical arthritic score (data not shown).
Sample preparation.
Single-cell suspensions of lung and spleen were prepared after collagenase/DNase digestion, as described elsewhere [20, 21]. Fractions were serially diluted and plated, to determine bacterial load. Total viable cell counts, determined by trypan blue exclusion, were used in conjunction with flow-cytometric data, to determine absolute numbers of cell populations. Before intracellular cytokine analysis, samples were incubated for 5 h in Ultra-Low cluster plates (Costar Corning) with 5 g/mL Brefeldin A (Sigma).
Flow cytometry.
MAbs used were phycoerythrin, fluorescein isothiocyanate, allophycocyanin (APC), and biotin conjugates. Biotinylated MAbs were used in conjunction with streptavidin-APC (BD Pharmingen). The following clones were used (all from BD Pharmingen, unless stated): 145.2C11 (CD3), GK1.5 (CD4), 536.7 (CD8), HL3 (CD11c), RA3.6B2 (B220), RB68C5 (Ly6G/Ly6C), F4/80 (macrophage), 2G9 (MHC-II), H57597 (TCR ), XT22 (TNF), C15.6 (IL-12p40/70), JES5-16E3 (IL-10), XMG1.2 (interferon ), 11B11 (IL-4), FA-11 (CD68; Serotec), and isotype control MAbs R334 (rat IgG1), R35-95 (rat IgG2a), and A95-1 (rat IgG2b).
Flow-cytometric analysis was conducted as described elsewhere [21]. Data were acquired by use of a FACSCalibur flow cytometer and were analyzed by use of CellQuest Pro 4.0.2 software (Becton Dickinson, for both).
Histopathological examination.
Organs were fixed in 2% formaldehyde, mounted in paraffin, and cut and stained as required before assessment and photography by use of a Zeiss Axioplan microscope (Zeiss) and Magnafire CCD camera (Optronics).
RESULTS
Noncritical polymorphonuclear leukocyte involvement after nonlethal challenge.
DBA/1 mice are acutely susceptible to S. pneumoniae infection. After inl challenge with 1 × 105 S. pneumoniae serotype 3, death occurred within 7296 h, despite a significant, rapid local phagocyte response (authors' unpublished data). To examine in detail the pulmonary immune responses to S. pneumoniae infection in the nonimmunocompromised host, an alternative model of invasive infection, with pulmonary inflammation and histological signs of pneumonia followed by bacterial clearance and host survival, was established by use of S. pneumoniae serotype 6B. After inl infection of DBA/1 mice with 1 × 1081 × 109 cfu of serotype 6B, as determined by serial-dilution plating, the majority of bacteria were rapidly cleared, with none detectable in lungs at day 7. Bacteremia peaked at day 1 (mean ± SD, 4.7 × 103 ± 3.4 × 103 cfu/mL; bacteria were detectable in 7 of 9 mice), whereas pneumococci in the spleen peaked at 48 h (mean ± SD, 3.5 × 103 ± 2.1 × 103 cfu). No viable pneumococci were recovered from the blood after day 3 or from the spleen after day 5. No differences were observed in the relative rate of bacterial clearance over an inl dose range of 1 × 1071 × 109 cfu (data not shown).
Infection resulted in a >2.5-fold increase in the total number of viable cells recovered from lungs as early as 1824 h after infection (figure 1A), peaking at day 2 but remaining significantly elevated at day 10 after infection (P < .01). Increased cellularity corresponded to a >30-fold increase in F4/80-CD68-Gr1HighCD11bHigh PMNs (figure 1B and 1C). To assess the functional consequences of PMN infiltration with respect to bacterial clearance, mice were treated ip with 0.5 mg of RB6-8C5, an MAb that depletes PMNs [22] (and plasmacytoid dendritic cells ; data not shown [23]), 24 h before S. pneumoniae challenge. RB6-8C5mediated depletion resulted in a >98% loss of PMNs in all organs analyzed (data not shown). PMN-depleted mice had an 10-fold higher mean lung bacterial load at day 1, compared with that in untreated control mice (mean ± SD, 4.5 ± 2.8 × 107 vs. 3.4 ± 2.7 × 106 cfu), but had a mean load that was equivalent to that in control mice at day 3 (mean ± SD, 7.2 ± 1.6 × 104 vs. 4.5 ± 4.0 × 104 cfu) and had complete clearance by day 7. Therefore, although PMNs are involved in early lung responses, their presence and TNF production are not essential for primary resistance to pneumococcal infection.
Mononuclear phagocyte responses.
PMNs are present at very low numbers in the normal lung, compared with F4/80+CD68+Gr1Low mononuclear phagocytes (figure 1B1D). These latter cells increased in number during nonlethal infection with serotype 6B, although less rapidly than did PMNs. Macrophage-recruitment kinetics after S. pneumoniae 6B challenge appeared to be bimodal; there was a primary influx that peaked at day 2 and a secondary influx that peaked at day 5 (figure 1D). By day 5, the number of pulmonary macrophages had increased >5-fold, compared with that in control mice, and remained significantly elevated at day 10 (although the number was reduced, compared with that at day 5; P < .0002).
In naive mice, TNF expression by pulmonary PMNs was negligible. During infection, a small but significant proportion of PMNs (0.5%2%) actively secreted TNF (figure 2A). In contrast, 13% of pulmonary macrophages constitutively expressed TNF in naive mice. At 24 h after inl challenge with serotype 6B, TNF-expressing macrophages were increased in number by >5-fold, although only 27% of macrophages expressed TNF at this time. At 48 h, only 14% expressed TNF, although the absolute number of TNF-expressing macrophages in the lung was still increased, compared with that in control mice (figure 2A). In contrast to PMNs, pulmonary macrophages in infected mice expressed IL-12, albeit at a low frequency (figure 2B). Macrophage IL-12 expression was sustained through day 7. Minimal numbers of IL-10expressing macrophages were observed throughout the course of infection (data not shown). No significant expression of IFN-, TNF, IL-12, or IL-10 by other lung cell populations (including T cells, B cells, and DCs) was observed at any time (data not shown). These data identify, for the first time, the major cellular source of TNF and IL-12 in the S. pneumoniaeinfected lung.
TNF is not an essential mediator of the primary cellular antipneumococcal response.
It has been suggested that TNF plays a critical role in primary immunity to S. pneumoniae. To address the role played by TNF in the cellular response to S. pneumoniae serotype 6B infection, DBA/1 mice were treated with either monospecific anti-TNF (cV1q [17]) or isotype control MAb (cVam) before inl infection. We then examined all parameters of the wild-type (wt) pulmonary response, described above, in control MAb- and cVIq-treated mice. Strikingly, we found no significant difference between these groups of mice in any of the parameters measured. There was no impact on the capacity of treated mice to clear pulmonary or systemic bacterial loads (figure 4 and data not shown). Phagocyte responses progressed similarly, with similar neutrophil and macrophage involvement, and the loss of pulDCInt occurred with similar kinetics and magnitude (figure 4). Similarly, mice treated with TNFR-Ig fusion protein, which inhibits both TNF and lymphotoxin- function, did not alter host resistance or the nature of inflammatory responses (data not shown). Neither administration of cIVq nor TNFR-Ig resulted in any change in IL-12, IFN-, IL-10, or IL-4 expression, compared with that in control mice (data not shown).
To confirm that primary resistance and regulation of pulmonary inflammatory responses to S. pneumoniae serotype 6B infection were TNF independent, we challenged B6 mice and TNF-/- mice on a B6 background. S. pneumoniae 6B inl challenge was not fatal in either mouse strain, and the kinetics of bacterial clearance in TNF-/- mice were almost indistinguishable from that in wt mice. The mean ± SD lung bacterial loads in TNF-/- mice were 3.3 ± 1.8 × 106 cfu at day 1 (compared with 3.2 ± 1.8 × 106 cfu in B6 mice) and 0.9 ± 0.8 × 105 cfu at day 3 (compared with 0.7 ± 1.8 × 105 cfu in B6 mice) of infection. Two of 6 mice in both groups had pneumococci in their lungs at day 7 (mean, <300 cfu), with none recoverable at day 10. The number of bacteria recovered from the spleen and blood of infected TNF-/- mice did not significantly exceed that recovered from the spleen and blood of wt mice at any time (data not shown).
TNF deficiency exacerbates liver damage during disseminated S. pneumoniae serotype 3 infection.
To determine whether TNF has a further, potentially critical role in the response to virulent S. pneumoniae infection, anti-TNFtreated mice, control MAb-treated mice, and TNF-/- mice were challenged inl with 1 × 105 cfu of serotype 3 bacteria. wt B6 mice, as noted above for DBA/1 mice, died of inl serotype 3 infection within 7296 h. To examine the role played by TNF during this infection, all mice were killed at 48 h after challenge, at which time point the control mice exhibited 100% survival, compared with 70% in the anti-TNFtreated mice (2 = 3.53; P = .03) and 40% in the TNF-/- mice (2 = 8.57; P = .002). These results are comparable with those of previous studies [57]. Mean blood bacterial load was comparable in control and anti-TNFtreated mice but was significantly increased in TNF-/- mice (mean ± SD, 71.5 ± 16.7 × 106 cfu/mL; P = .04, vs. control mice); a similar pattern was observed in the lung (figure 5 and data not shown).
Despite increased local and systemic bacterial loads resulting from TNF deficiency, the cause of death and the role played by TNF remained undefined. In control mice, serotype 3 infection was primarily associated with pulmonary neutrophil influx and increased pulDCInt numbers 48 h after challenge (figure 5). The quantitatively reduced PMN responses associated with serotype 3 infection, compared with those associated with serotype 6B infection, were likely due to the comparatively low infective dose (105 cfu). However, acute, lethal S. pneumoniae infection induced pulDCInt accumulation rather than the pulDC loss observed after nonlethal S. pneumoniae challenge (figures 3 and 4). Although TNF blockade did not affect the total cellular response at this time point, a significant increase in neutrophil number and a significant decrease in pulDC number was observed in anti-TNFtreated mice (figure 5). Exacerbation of these effects was associated with total TNF deficiency (data not shown).
To assess systemic influences of TNF deficiency in lethal infection, lung and liver samples were evaluated histopathologically. Lung infiltration was equivalent in each case; control MAb- and anti-TNFtreated mice exhibited massive leukocytic infiltration, with apparent areas of consolidation (figure 6A6C). Liver sections from control MAb- and anti-TNFtreated mice showed signs of systemic disease activity, although these groups were visually comparable (figure 6D and 6E). However, in TNF-/- mice, these signs were exacerbated, with extensive hepatocyte and Kupffer cell vacuolation, prominent hepatocyte nucleoli, and proteinaceous sinus content (figure 6F). Together, these data suggest a role, distinct from any contribution to antibacterial activity, for TNF during S. pneumoniae serotype 3 infection in prolonging host resistance to systemic organ pathology resulting from critically disseminated infection.
DISCUSSION
S. pneumoniae is a common causative agent of pneumonia and invasive pneumococcal disease [3] and is frequently associated with antibiotic resistance [2], making it of high clinical relevance. Host resistance to S. pneumoniae has been associated with TNF activity in both murine models [57, 1012] and human clinical trials [25, 26]. How TNF exerts its host-protective effects has not previously been addressed. We used both nonlethal and lethal challenges to examine in detail the role played by TNF in antiS. pneumoniae responses. The nonlethal serotype 6B model illustrated previously undefined cellular features of the primary pulmonary response to pneumococcal infection and clearly demonstrated that equivalent and protective antipneumococcal immune responses occur in the complete absence of TNF. In addition, a role for TNF in protection from systemic pathology after serotype 3 challenge was identified.
For nonlethal S. pneumoniae infection, our data demonstrate that, although neutrophils are the predominant phagocyte, neutrophilic influx is not essential for bacterial control, confirming similar observations from a cyclophosphamide-induced leukopenia model [27]. Even large inocula of serotype 6B pneumococci were readily cleared from neutrophil-depleted mice. Surprisingly, despite the large bacterial burden, few neutrophils expressed inflammatory cytokines such as TNF. Previous studies of the impact of the neutralization of TNF have suggested differences in PMN availability that we did not observe here [7, 10]. Indeed, PMN depletion had little impact on bacterial clearance in the high-dose, nonlethal model.
In contrast to the neutrophil response, an apparently bimodal macrophage response to serotype 6B infection occurred, which was suggestive of 2 waves of recruitment. During early infection, macrophages are likely involved in bacterial phagocytosis and killing and in amplification of responses through the production of inflammatory mediators. At later time points, a further influx of macrophages may be required to dispose of large numbers of apoptotic neutrophils, as has been observed after acute lipopolysaccharide-mediated inflammation [28].
Bacterial evasion of phagocytosis, in particular by macrophages, via virulence factors (including the release of substantial amounts of capsular polysaccharide) may be critical in the development from contained (pulmonary) infection to potentially fatal disseminated (bacteremic) infection. Correlation between mortality and pneumococcal capsule concentration in the lung was shown in humans as early as 1942 [29] and has been confirmed in animal models [13, 30, 31]. Together with the present data, these studies indicate that macrophages are the critical phagocyte for antiS. pneumoniae immunity [32].
We have identified that, as after antigen exposure [33] or pulmonary viral infection [34, 35], DCs are rapidly lost from the lung in response to nonlethal S. pneumoniae infection. It is probable that pulDCs migrate to draining lymph nodes after challenge, although S. pneumoniaemediated DC apoptosis [36] cannot be excluded as a mechanism that results in reduced pulDC numbers. Further studies that involve in vivo tracking and functional analysis will determine the fate of pulDCs and the role they play in the development of acquired immunity to S. pneumoniae. DCs initiate humoral responses to both protein and polysaccharide pneumococcal antigens [37], and bloodborne pre-DCs may carry bacteria directly to the splenic marginal zone [38]. Nevertheless, DCs are unlikely to play any significant role in primary clearance of pneumococci from the lung or bloodstream.
Most importantly, our data (from both the nonlethal and lethal models) argue strongly against an indispensable role for TNF in pulmonary responses to S. pneumoniae infection. Rather, TNF deficiency had no significant impact on local or systemic bacterial clearance or on multiple cellular criteria of inflammation after challenge with an invasive but controllable pneumococcal infection. Lethal pneumococcal challenge induced a distinct pulmonary response that had similarities to (rapid, PMN-dominated cellular influx) and differences (increased pulDC number) from nonlethal infection. In this case, TNF may be involved in regulating the extent, rather than the occurrence, of these pulmonary cellular responses. However, in the present study, clear and extensive signs of liver damage were associated only with TNF deficiency. TNF is clearly involved in the regulation of fatal infection with highly virulent S. pneumoniae serotypes, as shown here and previously [57, 1012]. The present study further demonstrates that a significant consequence of TNF deficiency during systemic infection is an exacerbation of hepatic breakdown.
Severe TNF deficiency predisposes mice to death after infection with a range of pathogens, with little correlation to pathogen load or even pathogen viability [39]. Therefore, a critical function of TNF in models of virulent S. pneumoniae infection may be as an amplifier of mechanism(s) that protect against systemic organ failure [40], possibly through enhancing anti-inflammatory cytokine production (e.g., IL-10 production) [41]. Serious pneumococcal infections, including those described in previous models, involve substantial bacteremia (i.e., pneumococcal sepsis). At best, TNF blockade during human sepsis has no effect on outcome [42, 43]; at worst, it significantly increases mortality [44].
Our data, therefore, suggest a compartmentalized necessity for TNF during S. pneumoniae infection. Pulmonary cellular antipneumococcal responses progress essentially independently of TNF activity. Although the final cause of death during disseminated S. pneumoniae infection remains undefined, it is likely a combination of bacterial load, lung inflammation, and systemic organ failure. It is this latter complication of infection, rather than antibacterial mechanisms, to which TNF appears to make its most significant protective contribution.
Acknowledgments
We thank Colleen Marano (Centocor, Malvern, PA), for generous provision of reagents cV1q, cVam, and tumor necrosis factor receptor immunoglobulin and for helpful comments; David Katz (University College London, London, UK) and Alero Thomas, for advice on histology; Richard Williams (Kennedy Institute of Rheumatology, London, UK), for assistance with the collagen-induced arthritis model; Lamine Mbow, Pat Geraghty, and Bernie Scallon (Centocor) and Marc Feldmann (Kennedy Institute of Rheumatology), for helpful comments on the manuscript; and the staff of the Biological Service Facility, London School of Hygiene and Tropical Medicine, for animal husbandry.
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