The activity of innate immunity is not simply dictated by the presence of an antigen but also by the balance between negative regulatory and immune potentiator pathways. Even in the absence of antigen, innate immunity can ‘inflame’ if negative regulators are absent. This resting state is adaptable and dictated by environmental influences, host genetics and past infection history. A return to homoeostasis post inflammation may therefore not leave the tissue in an identical state to that prior to the inflammatory event. This adaptability makes us all unique and also explains the variable outcome experienced by a diverse population to the same inflammatory stimulus. Using murine models we have identified that influenza virus causes a long-term modification of the lung microenvironment by a de-sensitization to bacterial products and an increase in the myeloid negative regulator CD200R (CD200 receptor). These two events prevent subsequent inflammatory damage while the lung is healing, but also they may predispose to bacterial colonization of the lower respiratory tract should regulatory mechanisms overshoot. In the extreme, this leads to bacterial pneumonia, sepsis and death. A deeper understanding of the consequences arising from innate immune cell alteration during influenza infection and the subsequent development of bacterial complications has important implications for future drug development.
Influenza infection and secondary bacterial pneumonia
Influenza infection causes considerable morbidity and mortality both seasonally and in sporadic pandemic infections . In the 1918/1919 influenza pandemic, several million deaths were recorded  and, although accurate figures are unavailable, it is apparent that many of these cases were complicated by the presence of a secondary bacterial infection, which may well have been the ultimate cause of death . Indeed, a significant number of these fatalities may have been caused by multiple concurrent bacterial species. Even during seasonal influenza infection, 4–14 days after resolution a recurrence of fever, dyspnoea, productive cough and pulmonary consolidation often arises as the result of bacterial superinfection, most commonly caused by Streptococcus pneumoniae, Staphylococcus aureus and Haemophilus influenzae. However, not all patients suffer to the same extent and, although genetic and environmental differences contribute to these differences, our past infection history also leaves a ‘fingerprint’ that is just as unique and affects our responses to future infections. Figure 1 outlines the immunological sequence of events following influenza infection and highlights some of the changes to the lung environment associated with susceptibility to secondary bacterial infection.
The sequence of events during respiratory viral infection and the timing of secondary bacterial infection
Enhanced susceptibility to secondary bacterial complications following influenza can be partly explained by the mode of viral replication. Influenza is a cytolytic virus that causes necrosis and desquamation of bronchial epithelial cells, in severe cases down to the basal layer. Hyaline membrane formation in alveolar ducts and alveoli and evidence of epithelial repair are also prominent features of disease . As a result, barrier defence in the lung is compromised and bacteria may colonize the lung more easily and have easier access to the bloodstream, from whence they can cause systemic disease. Another possible explanation is that influenza infection activates cells in the lung causing them to up-regulate a variety of molecules on their surface, some of which could potentially be used by bacteria to adhere to and infect cells. A further consequence of influenza infection is the apoptosis of inflammatory cells in the lung. A recent study suggests that engulfment of apoptotic cells (a process known as efferocytosis) enhances the production of IL-10 (interleukin-10) and TGF-β (transforming growth factor β) and renders the lung susceptible to infection with S. pneumoniae . Dendritic cells are similarly affected although in this case the process appears to be mediated by nitric oxide . Apoptotic cell clearance by efferocytosis may explain why IL-10 concentrations are 50 times higher after influenza infection and also why IL-10 neutralization prevents bacterial outgrowth in influenza-infected mice . Even inflammatory cytokines such as IFN-γ (interferon-γ), which dominates during experimental influenza infection, decrease the antibacterial capacity of alveolar macrophages . We have identified another significant influence of influenza: a desensitization of airway macrophages to bacterial TLR (Toll-like receptor) agonists . This alteration is robust and long-lasting and suggests that in addition to adaptive, lymphocyte-based immunological memory, innate immune cells in the lung are altered after infection and that this ‘education’ of innate immunity can affect responses to future infections. These significant alterations in key antibacterial immune cells are also likely to affect soluble mediators important for microbial control; alveolar macrophages and airway epithelial cells, for example, contribute to the antibacterial peptide landscape [9,10] and a shift in cellular dynamics during infection will have significant ramifications for this first line of defence.
CD200 in lung respiratory innate immune homoeostasis
It is becoming clear that innate immunity requires constant restraint in the absence of pathogenic micro-organisms and that a disruption of homoeostatic mechanisms may lead to mild or chronic inflammation. This appears to be true of the lung where in homoeostasis, lung macrophages dominate but are suppressed by the local microenvironment. CD200R (CD200 receptor), for example, transmits a negative regulatory signal to cells of the myeloid lineage [11–15] and is expressed at unusually high levels on airway macrophages owing to high local concentrations of IL-10 and TGF-β lodged on, or secreted by, the respiratory epithelium [16–18]. The CD200R ligand, CD200, is also expressed on the luminal aspect of the airway epithelium and limits alveolar macrophage activity. This tightly controlled negative regulation is overcome during influenza infection, probably when the respiratory epithelium is lysed as described above. Furthermore, alveolar macrophages up-regulate a diverse array of immune potentiators during inflammation such as TLRs and the immune potentiator OX40L (OX40 ligand; the ligand for the late T-cell co-stimulator OX40). In the presence of antigen, OX40L-expressing cells deliver a signal to activated OX40+ T-cells that increases the expression of cytokine receptors [19,20] as well as promoting the transcription of anti-apoptotic factors such as Bcl-2 family members . Reverse signalling through OX40L on the antigen-presenting cell can also increase inflammatory cytokine production . This combination of a loss of negative signals and the increased expression of pro-inflammatory immune potentiators tips the balance from regulation to inflammation, overcoming homoeostatic control mechanisms to remove invading pathogens. This scenario may also apply in the gastrointestinal tract where CD200R is also expressed on macrophages  and IL-10 and TGF-β levels are high . In contrast, splenic macrophages express low levels of CD200R, consistent with the low expression of IL-10 and TGF-β in this site. As such, the threshold of responsiveness of alveolar and gastrointestinal macrophages, to the same inflammatory stimulus, is higher than splenic or lymph node macrophages. This is clearly beneficial in these environmentally exposed sites but if regulation is overly restrictive, pathogens may escape detection.
Many of the molecules involved in homoeostasis also feature in inflammatory resolution. However, in some individuals or after certain infections, these processes may overshoot, leaving the host susceptible to other secondary infections. After influenza infection, alveolar macrophages express much higher levels of the regulatory molecule CD200R  and IL-10 concentrations are greater, probably contributing to the reduced responsiveness to subsequent bacterial infection described in our earlier work.
Should we be using antibiotics for influenza-induced illness?
Inhibition of regulatory molecules during the resolution phase of influenza infection may prevent bacterial infection, but neutralization of homoeostatic pathways such as IL-10 and TGF-β is costly and not without inflammatory consequences. It may therefore be beneficial to apply antibiotics during the resolution phase of influenza infection. McCullers and co-workers have recently shown that the choice of antibiotics is critical to realize an improved outcome and depends on whether or not the antibiotic lyses bacterial species . Ampicillin is a β-lactam antibiotic that clears S. pneumoniae by bacterial lysis, but, when administered to influenza-co-infected mice, amplifies inflammatory cytokines and mortality. Bacterial lysis during an already highly inflammatory response therefore contributes to, rather than alleviates, the disease. Clinidamycin, however, is a protein synthesis inhibitor that does not lyse bacteria and such treatment in influenza/S. pneumoniae-co-infected mice produces less TNF (tumour necrosis factor), reduces weight loss and all mice survive despite incomplete clearance of the bacteria. Bacterial lysis also liberates a number of other immune-stimulatory proteins that contribute to inflammatory disease, so combining anti-inflammatory properties with protein synthesis inhibition in the form of Azithromycin produces an even greater benefit .
Conclusions and future implications
These and other studies suggest that, rather than existing in a natural state of quiescence, macrophages in the lung must be actively suppressed by other tissue-resident cells to prevent an immune response from developing. To break through this suppression, macrophages must receive a number of signals that indicate a pathogenic presence. This level of control prevents the formation of immune responses towards innocuous inhalants and is especially important in the lungs, which are bombarded by foreign particles with every breath, but which must avoid the damage associated with an immune response to maintain efficient gaseous exchange. After an infection, the lung needs time to repair and regenerate damaged tissue. The dampening of inflammation and an increase in the suppressive signals to macrophages and other innate immune cells may provide a chance for this repair to occur. However, the reduced activity also provides a window of opportunity for bacteria and other pathogens to infect. Processes involved in immune homoeostasis and inflammatory resolution are also likely to be disrupted in more chronic conditions such as chronic obstructive pulmonary disease and severe asthma. These alterations may contribute to disease pathogenesis but also leave the host susceptible to opportunistic infections in patients with these conditions.
It is clear that our infection history can have long-lasting implications in our future ability to combat pathogens and can also have an impact on autoimmunity. As we move into the era of personalized medicine, it is becoming increasingly important to take into account past infections and the development of co-infection models is one step towards understanding the ways in which infection history has an impact on disease.
Biochemical Basis of Respiratory Disease: Biochemical Society Focused Meeting held at AstraZeneca, Loughborough, U.K., 5–6 March 2009. Organized and Edited by Colin Bingle (Sheffield, U.K.) and Alan Wallace (AstraZeneca, U.K.).
This work was supported by the Medical Research Council [grant number P171/03/C1/048] and the National Institutes of health [grant number NGA: 1 UO1 AI070232-01].