The innate immune response to lower respiratory tract infections has been studied largely from the perspective of the local lung microenvironment, with remarkably little known about extrapulmonary events that may influence disease outcome. Our present findings help elucidate the kinetics, magnitude, and mechanisms of the hepatic APR during bacterial pneumonia. The observed liver response to lung infections included not only the expression of APPs, the hallmark of the APR, but also the activation of the transcription factors STAT3 and RelA. These transcription factors were both essential to APP induction by hepatocytes stimulated in vitro with cytokines. Finally, our results unequivocally confirm the importance of the cytokines TNF-α, IL-1, and IL-6 in promoting the APR, since loss of these signaling pathways had profound effects both on hepatic APP expression and on transcription factor activity in vivo. These findings highlight a communication axis between the lung and liver during pneumonia.
In order to more definitively analyze the APR during pneumonia, we performed every experiment with two different pathogens, E. coli
and S. pneumoniae
, both of which are relevant to patients diagnosed with lung infections (1
). These two bacteria elicit very different responses in the lungs, with different requirements for host defense mediators and distinct mechanisms of virulence and pathophysiology. In the context of the APR, an important difference between these two organisms is the incidence of bacteremia, which is high during pneumococcal pneumonia but absent in response to our selected dose of intratracheal E. coli
; also unpublished observations). Consequently, initiation of the hepatic APR during E. coli
pneumonia in the present studies likely resulted from circulating factors produced directly in response to lung infection rather than from bacterial activation of hepatocytes secondary to bacteremia. In addition to circulating host-derived factors, however, it is plausible that disseminated S. pneumoniae
directly influences hepatic APP expression, since bacteremia can occur in response to lung infections with our selected serotype (type 3) of pneumococcus (10
; also unpublished observations).
Despite the different host responses elicited by E. coli
and S. pneumoniae
in the lungs, our present results identify IL-6, TNF-α, and IL-1 as critical factors linking pulmonary innate immunity to hepatic transcription factor activation and APP synthesis, regardless of the infectious stimulus. In the case of E. coli
lung infection, we have previously shown that loss of either IL-6 or early-response cytokine signaling significantly reduces acute pulmonary inflammation, despite relatively normal expression of neutrophil chemoattractants and other proinflammatory mediators in the lungs (20
). Activation of STAT3 and RelA is only modestly affected (STAT3) or unaffected (RelA) in the lungs of pneumonic mice lacking IL-6 or TNF-α/IL-1 signaling, respectively (20
). In comparison, loss of either cytokine pathway in the current study caused large decreases in the corresponding transcription factor activity and in APP expression in the liver, implicating extrapulmonary signaling as a major cytokine function during pneumonia. These results link TNF-α/IL-1-induced RelA signaling to liver transcription factor activity and APP synthesis, and they build upon our previous finding that livers from pneumonic TNFR1- and IL1R1-deficient mice have impaired NF-κB DNA binding in response to E. coli
). IL-6, TNF-α, and IL-1 are also critical for promoting acute inflammation and host defense against S. pneumoniae
in the lungs, for reasons that remain somewhat unclear, particularly in the case of IL-6 (21
). In agreement with our findings for E. coli
-challenged mice, these cytokines were absolutely critical for the APR in the liver during pneumococcal pneumonia, further evidencing a functional role for their presence outside of the lung. Taken together, these data support the postulate that a mechanism by which these cytokines contribute to local lung inflammation and host defense in the lungs may be the induction of APP transcription in the liver.
Our loss-of-function mouse models clearly indicated that IL-6, TNF-α, and IL-1 signaling is a critical liaison between the lungs and liver in response to both gram-negative and gram-positive pathogens. Interestingly, the expression of these cytokines by the liver appears to differ dramatically between these types of infection. During pneumococcal pneumonia, cytokine mRNA expression was increased in the lungs, with no detectable change in the liver, suggesting an endocrine lung-liver axis through which lung-derived cytokines beget a hepatic APR. In contrast, E. coli-induced lung infection caused rapid elevations in the levels of cytokine transcripts in both the lungs and the liver, suggesting that either tissue may be an important modulator of downstream APP expression. Elucidating the infection-specific mechanisms and functional significance of liver IL-6, TNF-α, and IL-1 expression is an important goal for future studies.
The prior understanding of mechanisms mediating APR induction during pneumonia has been fragmentary and speculative. Circulating levels of SAP and complement component 3 are significantly reduced in IL-6-deficient mice during pneumococcal pneumonia (42
). Our current results strongly suggest that this is due to loss of IL-6-induced STAT3 activity and APP mRNA expression in the liver. Hepatic APP expression in response to intrapulmonary lipopolysaccharide (LPS) is dependent on IL-6 (15
) but not on TNF-α (43
). This differs somewhat from the findings of the present study, which indicate that the hepatic APR during either of two different bacterial pneumonias requires IL-6 and also TNF-α or IL-1. One possibility to explain this difference is that the biological response to LPS in the lungs differs from that elicited by living bacteria. Alternatively, IL-1 signaling alone may be sufficient to make up for the absence of TNF-α, such that the requirements for these early-response cytokines (each of which activates NF-κB RelA) can be accurately determined only when signaling from both is eliminated. This concept is supported by multiple studies showing a compensatory relationship between TNF-α and IL-1 in response to pulmonary infection (21
STAT3 and RelA have previously been shown to mediate APP expression in vitro (6
). It has also been shown that STAT3 disruption in vivo limits APP expression in response to IL-6 or endotoxemia (4
). A limitation of our own in vivo results is that they do not indicate whether STAT3 and RelA are the actual means through which IL-6 and early-response cytokines, respectively, initiate hepatic APP synthesis during pneumonia. To causally link transcription factor activity to cytokine-induced APP expression, we employed an in vitro loss-of-function approach. siRNA-induced knockdown ablated STAT3 and RelA protein expression in the murine hepatocyte line AML12, and loss of either transcription factor prevented cytokine-induced SAA1 mRNA expression. These results suggest that reduced APP synthesis in IL-6−/−
and TM mice is a direct result of decreased STAT3 and RelA function. Notably, knockdown of either transcription factor equally decreased and nearly completely eliminated murine SAA1 expression, suggesting that each is essential. This finding supports those of previous promoter-reporter experiments with HepG2 human hepatoma cells, which indicate that human SAA promoter activity is dependent on both RelA and STAT3 (6
). Consistent with essential roles for STAT3 and RelA in integrated responses to microbes in the lungs, STAT3 mutation in humans causes hyper-immunoglobulin E syndrome, characterized by severe lung infections (13
), and RelA deficiency in mice renders them susceptible to severe lung infections (2
The effects of IL-6 and TNF-α/IL-1 deficiencies on the hepatic APR, coupled with the relatively smaller effects of these cytokines on transcription factor activity and other indices of inflammatory signaling in the lungs (20
), implicate the absence of lung-liver communication as one possible reason for impaired immunity in these cytokine-deficient mice. However, the true biological significance of the APR during bacterial pneumonia (or in any setting) is unclear (14
). Studies of individual APPs have identified relevant immunological roles for multiple APPs. For example, mice lacking a functional gene for LBP have reduced pulmonary inflammation and host defense function in response to intrapulmonary LPS and Klebsiella pneumoniae
, respectively (8
), and SAP-deficient mice have reduced pulmonary and systemic clearance of S. pneumoniae
). Complementarily, exogenous administration and/or overexpression of LBP, SAA, or CRP can be protective against bacterial infection (18
). Although these studies demonstrate that particular APPs benefit antibacterial host defense, they do not address the APR specifically or fully. The APR is defined by changes in APP levels compared to baseline rather than by the presence or absence of an APP. Because APP gene targeting eliminates even the often substantive baseline expression of that APP, this strategy elucidates functions of that protein but not the APR. In addition, the APR involves coordinated changes in hundreds of genes (46
). Thus, individually targeting select APPs elucidates at best a minuscule fraction of the APR. The true functional significance of the APR remains elusive, and its elucidation is an important research goal that will require new tools or novel approaches.
Based on our current results, we propose that the hepatic APR serves as a downstream and functionally relevant target of inflammatory cytokines synthesized during pneumonia. While the significance of the APR will need to be more specifically and fully defined with future studies, the present data suggest that the APR may be a central aspect of the host defense functions that have been ascribed to TNF-α, IL-1, RelA, IL-6, and STAT3 during pneumonia.