We have previously shown that excessive inflammation and pathology associated with RSV or Cryptococcus neoformans
infection is attenuated in mice previously infected with influenza virus (unpublished data) (2
). This does not depend on cross-reactive adaptive immunity, and occurs even 6 mo after the initial infection. To investigate the putative role of innate immunity in this process, we introduced the bacterial TLR5 agonist flagellin, which produces transient cytokine release and rapid recruitment of neutrophils and macrophages to the lungs (12
), to such “post-influenza” mice, leaving an interval of at least 4 wk. At this time, virus is not detectable in the lungs, the mice have recovered their initial starting weight, and total lung cellularity and proinflammatory cytokines have returned to preinfection levels (Fig. S1, available at http://www.jem.org/cgi/content/full/jem.20070891/DC1
). As previously reported (8
), minor populations of CD8+
T cells, CD11c+
cells, and isolated lymphoid aggregates were observed in the post-influenza lungs, whereas the overall architecture of the lungs was similar to noninfected animals (Fig. S2). Administration of the TLR5 ligand flagellin into the post-influenza lungs caused a significantly reduced early neutrophil transmigration into the airways compared with control mice (88.1 ± 8.3% reduction; n
= 16; P < 0.001; ), which was confirmed by immunohistology (unpublished data) and was not caused by a delay in the kinetics of recruitment (24-h time point; ). This effect was independent of the mouse genetic background (, C57BL/6 and BALB/c), and was evident even when the interval between influenza and flagellin was increased to 3 or 6 mo ().
Figure 1. Long-term impairment of TLR5-dependent neutrophil recruitment after resolution of an influenza infection. BALB/c (A and C) or C57BL/6 (A and B) mice were infected i.n. with 50 HA of influenza X31 (post-flu, filled bars) or PBS as control (ctrl, open bars), (more ...)
A similar impairment of neutrophil recruitment in the post-influenza airways was also observed with the TLR4 agonist LPS (), which is a major trigger of inflammation during Gram-negative bacterial infection and is often used as a model for acute lung injury. At 48 h, a reduction in macrophage recruitment was also observed (control, 5.8 ± 0.2 × 105; post-influenza, 3.4 ± 1.4 × 105; n = 4; P = 0.041), suggesting that general cell recruitment is affected in post-influenza lungs. Neutrophil recruitment to TLR2 ligation (LTA), which is associated with recognition of Gram-positive bacteria, also showed a modest reduction (). In addition, this effect was not observed with inactivated virus (unpublished data) and could be extended to the noncytopathic virus RSV (84.1 ± 10.7% reduction of neutrophils 6 h after flagellin challenge; n = 5; P = 0.028).
Figure 2. Reduced airway cell recruitment occurs after secondary bacterial challenge. Control (PBS-treated) or post-influenza mice (6 or 2 wk after influenza, as indicated) were inoculated with 1 μg LPS or 25 μg LTA (A) or were infected with 5 × (more ...)
Because desensitization to bacterial microbial-associated molecular patterns after resolution of influenza infection may explain why secondary bacterial infections occur, we next examined the response to the opportunistic Gram-negative bacterium Pseudomonas aeruginosa
because early containment of this organism is known to require TLR4 and TLR5 (13
). As observed with flagellin and LPS, neutrophil recruitment was reduced up to fourfold at 4 h and correlated with enhanced bacterial load ().
Similar results were obtained for the Gram-positive organism group B streptococcus. Airway cellularity was also reduced, and the bacterial burden was enhanced in the post-influenza lung (). Streptococcus pneumoniae
is a common pathogen in secondary pneumonia after influenza infection. Peak neutrophil recruitment was once again affected 6 wk after influenza infection, and uncontrolled bacterial replication led to death in 60% of mice compared with 17% in control animals (). In patients, secondary bacterial infections usually occur within 2 wk of influenza infection (4
). Reducing the time interval to 2 wk between the 2 infections produced the same effect in our model ().
Reduced neutrophilia to a second respiratory stimulus may reflect enhanced apoptosis within the lungs, an inability to traverse the epithelial layer, or a reduction of chemotactic signals to draw them there. We show it is the latter. Although some viral infections induce apoptosis of granulocytes during the acute phase of infection (15
), the proportion of apoptotic neutrophils was low and comparable between post-influenza (4.7 ± 1.4%) and control mice (4.8 ± 0.9%) 6 h after flagellin challenge (n
= 5). Impaired neutrophil transmigration, which is caused by possible modification of the lung microstructure after virus infection, was ruled out by showing equivalent numbers recruited to the airways of control and post-influenza mice after intranasal (i.n.) administration of the neutrophil chemoattractants, KC (CXCL1), and MIP-2α (CXCL2; ) (16
). However, flagellin-mediated induction of KC and MIP-2α () and proinflammatory cytokines () were reduced in the post-influenza lungs and airways compared with controls. We tested and confirmed that this impaired chemotactic signal was not caused by antimicrobial factors or neutralizing antibodies secreted in the airways that could potentially neutralize flagellin (unpublished data). In addition to protein, we show that KC and MIP-2α mRNA transcripts are reduced in post-influenza mice (). Altogether, these data indicate that the local induction of the TLR signaling pathway is altered by prior viral lung exposure.
Figure 3. The recruitment defect in post-influenza mice is associated with an impaired TLR-induced cytokine production. (A) Airway and lung neutrophil recruitment in post-influenza mice (2 mo after influenza) or control mice (2 mo after PBS treatment) were compared (more ...)
We next investigated which cell types were desensitized to TLR-mediated signals. AMs and radioresistant, nonhematopoietic resident cells such as epithelium are instrumental in inducing TLR-dependent early innate responses (13
). We identified that systemic (i.v.) administration of flagellin, which is likely to target lung endothelial cells (19
), did not impair neutrophil infiltration in the lung parenchyma or at peripheral sites ( and Fig. S3, available at http://www.jem.org/cgi/content/full/jem.20070891/DC1
), which suggests that alterations in airway apical TLR signaling is involved when flagellin is administered i.n. Therefore, we first investigated to what extent AMs were affected in post-influenza lungs. The reduction in cytokine production is not explained by reduced numbers () or an alteration in TLR levels (). AMs isolated 1 h after flagellin challenge and tested directly ex vivo displayed reduced mRNA transcripts for KC, MIP-2α, and TNF-α in post-influenza compared with control mice (), suggesting that transcriptional control of these genes is responsible for the reduced level of cytokines observed ().
Figure 4. Involvement of epithelial cells and AMs in post-influenza TLR desensitization in vivo. (A) AMs were isolated from post-influenza mice (6 wk after influenza) and control mice, and the level of TLR mRNA transcripts was assessed by real-time PCR. (B) Post-influenza (more ...)
Removal and analysis of cells ex vivo does not indicate whether transcriptional regulation occurs independently in this cell type or as a result of cooperation with others. Cross talk between AMs and alveolar epithelial cells (AECs) is known to occur in vivo (20
). To address this issue, we next individually isolated these populations from post-influenza mice and examined flagellin-induced NF-κB activation in vitro. Nuclear translocation of the p65 subunit of NF-κB in response to flagellin was inhibited in AMs () isolated from post-influenza mice, which was associated with a defect in chemokine production (). In contrast, flagellin-induced activation of NF-κB was not altered in post-influenza AECs (Fig. S4, available at http://www.jem.org/cgi/content/full/jem.20070891/DC1
). We therefore propose a model whereby sustained reduction of NF-κB activation in AMs after the initial viral infection leads to reduced inflammatory response and neutrophil recruitment. To support this hypothesis, we then conditionally depleted CD11c+
cells, including AMs, from post-influenza mice using CD11c–diphtheria toxin (DT) receptor (DTR) chimeric mice (21
). These mice were infected with influenza and depleted of CD11c+
cells using i.n. DT after resolution of infection (2 wk later). This resulted in an ~90% reduction in AMs (), as well as DCs and lung macrophages (Fig. S5), which is similar to that described by Landsman and Jung (23
). The pool of AMs was regained 3 wk later. In this scenario, the influence on neutrophil recruitment to subsequent flagellin challenge was lost (). Based on the dominant response occurring after apical (i.n.) administration of TLR agonists (), this data suggests that AMs are instrumental in the long-lived altered response in the post-influenza lung. This is further supported by the slow turnover of these cells in the airways (23
). However, we cannot exclude an indirect role for other CD11c-expressing populations, such as DCs and tissue macrophages. It remains to be determined whether AMs are directly affected by infection or their phenotypes are altered by interaction with recruited inflammatory cells. In addition, whether the pool of lung macrophages that give rise to AMs (23
) is also affected by previous infection warrants further investigation. Importantly, we are not describing simple TLR cross-tolerance, whereby TLR-activated cells are, for a short period of time, refractory to subsequent TLR stimulation (24
), as the effect persists for months after the initial infection. The molecular mechanisms responsible for long-term TLR desensitization remain to be resolved, and they may include up-regulation of intracellular antagonists such as IRAK-M, down-regulation of adaptor molecules, and/or sustained influence from other signaling pathways (25
Figure 5. Inhibition of TLR-induced NF-κB in AMs is responsible for reduced cell recruitment in post-influenza mice. (A) AMs isolated from control and post-influenza mice were stimulated for the indicated time with 1 μg/ml FliC and further stained (more ...)
Many mechanisms have been proposed to explain enhanced bacteria at the time of pandemic and seasonal influenza infection (4
) or RSV (26
), including a disruption of epithelial integrity, up-regulation of bacterial adhesion molecules, and/or an alteration in antibacterial peptides (27
). We now propose an additional mechanism, TLR desensitization, which is associated with reduced neutrophils and heightened secondary bacterial load. Furthermore, we are the first to examine long-term alterations of an innate immune pathway after resolution of respiratory viral infection. It is important to point out, however, that in addition to the reduced chemokine production, other TLR-dependent antimicrobial mechanisms, such as the production of microbicidal products, may also play a role.
The pertinent question is why would the lung leave itself vulnerable to bacterial infection in this way? In certain compartments, such as the lung, inflammatory cascades need to be regulated to prevent bystander tissue damage, which itself can be life threatening to the host. The lung, although it contains a heavy microbial load in the upper respiratory tract, is essentially sterile below the larynx, and it is devoid of any significant resident organized lymphoid tissue. Pathogens bypassing passive antimicrobial strategies can cause significant indirect pathology in the lungs because of the recruitment of excessive numbers of immune cells that occlude the airspaces. An alteration in responsiveness to TLRs is therefore beneficial for those infections associated with excessive immunity, but at the expense of other pathogens, such as bacteria. This phenomenon, although detrimental in a subpopulation of coinfected individuals, is likely to represent an evolutionary advantage for the respiratory tract and shares interesting similarities with immunosuppressive mechanisms operating at other mucosal sites.
In the gut, the microbial flora and associated TLR ligands play an essential role in mucosal homeostasis by actively inhibiting intestinal innate responses (28
). Furthermore, responsiveness of intestinal epithelial cells to TLR activation is impaired immediately after birth by exposure to exogenous endotoxin (29
). Although the distal lung cannot afford a bacterial flora, repetitive exposure to microbial products may induce a similar beneficial hyporesponsiveness. Associated with attenuated acute inflammation, lung dendritic cells in post-influenza mice are more efficient at presenting antigens and at promoting T cell responses (8
). Therefore, by reducing excessive inflammation and improving its ability to induce a specific adaptive response, the “experienced” lungs are better equipped to fight a secondary infection. The unfortunate consequence of attenuated TLR responsiveness may be susceptibility to bacterial infection. Although our studies investigate interactions between successive respiratory pathogens, similar effects may also occur in the lung to more soluble antigens, such as allergens.