A role for heat shock factor 1 in hypercapnia-induced inhibition of inflammatory cytokine expression

Ziyan Lu

S Casalino-Matsuda

Aisha Nair

Anja Buchbinder

G Budinger

Peter Sporn

Khalilah Gates

^{Department of Medicine, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA;}

^{Jesse Brown Veteran Affairs Medical Center, Chicago, Illinois, USA;}

^{Universities of Giessen and Marburg Lung Center, Giessen, Germany}

^{Correspondence: Northwestern University, Feinberg School of Medicine, 240 E. Huron, McGaw M-300, Chicago, IL 60611, USA., E-mail:}ude.nretsewhtron@setag-k

Received 2017 Dec 4; Accepted 2018 Jan 22.

Abstract

Hypercapnia, elevated levels of CO₂ in the blood, is a known marker for poor clinical prognosis and is associated with increased mortality in patients hospitalized with both bacterial and viral pneumonias. Although studies have established a connection between elevated CO₂ levels and poor pneumonia outcomes, a mechanistic basis of this association has not yet been established. We previously reported that hypercapnia inhibits expression of key NF-κB–regulated, innate immune cytokines, TNF-α, and IL-6, in LPS-stimulated macrophages in vitro and in mice during Pseudomonas pneumonia. The transcription factor heat shock factor 1 (HSF1) is important in maintaining proteostasis during stress and has been shown to negatively regulate NF-κB activity. In this study, we tested the hypothesis that HSF1 activation in response to hypercapnia results in attenuated NF-κB–regulated gene expression. We found that hypercapnia induced the protein expression and nuclear accumulation of HSF1 in primary murine alveolar macrophages and in an alveolar macrophage cell line (MH-S). In MH-S cells treated with short interfering RNA targeting Hsf1, LPS-induced IL-6 and TNF-α release were elevated during exposure to hypercapnia. Pseudomonas-infected Hsf1^+/+ (wild-type) mice, maintained in a hypercapnic environment, showed lower levels of IL-6 and TNF-α in bronchoalveolar lavage fluid and IL-1β in lung tissue than did infected mice maintained in room air. In contrast, infected Hsf1^+/− mice exposed to either hypercapnia or room air had similarly elevated levels of those cytokines. These results suggest that hypercapnia-mediated inhibition of NF-κB cytokine production is dependent on HSF1 expression and/or activation.—Lu, Z., Casalino-Matsuda, S. M., Nair, A., Buchbinder, A., Budinger, G. R. S., Sporn, P. H. S., Gates, K. L. A role for heat shock factor 1 in hypercapnia-induced inhibition of inflammatory cytokine expression.

Keywords: rodent, macrophage, bacterial infections, stress response, lung

Abstract

Hypercapnia, elevated levels of CO₂ in the blood, has been recognized as a marker of poor prognosis in patients with chronic and/or severe lung diseases, such as chronic obstructive pulmonary disease and acute respiratory distress syndrome (1 –7). Elevated CO₂ levels are also associated with increased mortality in patients hospitalized with community-acquired bacterial and viral pneumonias (8, 9). Although clinical studies have established a connection between elevated CO₂ levels and poor patient outcomes, they have not yet revealed the mechanistic basis for that association. Work from our laboratory and others, using both cell culture systems and in vivo animal models, supports the hypothesis that exposure to hypercapnia impairs the host’s innate immune response and host defense (10 –12).

In human and murine macrophages, we reported that hypercapnia attenuates the LPS-induced expression of TNF-α and IL-6– and NF-κB–regulated cytokines important for host defense in a pH-independent manner (11). We further showed that hypercapnia suppresses IL-6 and TNF-α expression in the lungs of mice infected with Pseudomonas aeruginosa, which is associated with increased mortality and impaired bacterial clearance (10). Interestingly, decreased expression of TNF-α, IL-6, and other NF-κB–regulated cytokines is also observed in macrophages exposed to “heat shock,” a modest increase in temperature of 1–2°C (13 –16).

Heat shock induces expression of heat shock proteins (HSPs), such as HSP70, which function as molecular chaperones that maintain proteostasis within the cell during stress-invoking stimuli, such as bacterial infections (17, 18). Induction of HSPs is a highly conserved response regulated by the transcription factor heat shock factor 1 (HSF1), which binds to heat shock elements in promoter regions of target genes (15, 16, 19). HSF1 has been shown to inhibit gene transcription of TNF-α, IL-6, and IL-1β, at least in part, by binding to heat shock elements in the promoter regions of those non–heat shock genes, highlighting the function of HSF1 as an integrator of stress responses in cells (16, 19, 20).

In this study, we show that hypercapnia activates HSF1 in murine macrophages. Knockdown of Hsf1 in a murine alveolar macrophage–like cell line (MH-S) prevents the hypercapnia-mediated inhibition of LPS-induced IL-6 and TNF-α expression. Consistent with these findings, we show that mice with reduced HSF1 expression (Hsf1^+/−) are protected from hypercapnia-induced inhibition of IL-6, TNF-α, and IL-1β during Pseudomonas pneumonia. Taken together, these results suggest a previously unrecognized role for HSF1 as a mediator of CO₂ signaling in LPS-stimulated murine macrophages and in the Pseudomonas-infected mouse lung.

n.s., not significant.

ACKNOWLEDGMENTS

The authors thank Dr. Karen Ridge and Dr. Navdeep Chandel (both from Northwestern University) for advice, reagents, and technical assistance. The authors acknowledge support from the U.S. National Institutes of Health (NIH) National Heart, Lung, and Blood Institute (Grants K01 HL108860, R01 HL131745, and P01 HL071643), and NIH National Institute on Aging (Grant P01 AG049665). The authors declare no conflicts of interest.

ACKNOWLEDGMENTS

Glossary

ad-Luc1	adenoviral vector encoding destabilized luciferase
BAL	bronchoalveolar lavage
HC	hypercapnia
HSF1	heat shock transcription factor 1
HSP	heat shock protein
IF	immunofluorescence
LDH	lactate dehydrogenase
NC	normocapnia
P_aCO₂	partial pressure of carbon dioxide in arterial blood
siRNA	short interfering RNA

Glossary

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