Intracellular heat shock protein-70 negatively regulates TLR4 signaling in the newborn intestinal epithelium

Amin Afrazi, Chhinder Sodhi, Misty Good, Hongpeng Jia, Richard Siggers, Ibrahim Yazji, Congrong Ma, Matthew D. Neal, Thomas Prindle, Zachary S. Grant, Maria F. Branca, John Ozolek, Eugene B. Chang, David Hackam

Research output: Contribution to journalArticle

Abstract

Necrotizing enterocolitis (NEC) is the leading cause of gastrointestinal-related mortality in premature infants, and it develops under conditions of exaggerated TLR4 signaling in the newborn intestinal epithelium. Because NEC does not develop spontaneously, despite the presence of seemingly tonic stimulation of intestinal TLR4, we hypothesized that mechanisms must exist to constrain TLR4 signaling that become diminished during NEC pathogenesis and focused on the intracellular stress response protein and chaperone heat shock protein-70 (Hsp70). We demonstrate that the induction of intracellular Hsp70 in enterocytes dramatically reduced TLR4 signaling, as assessed by LPS-induced NF-κB translocation, cytokine expression, and apoptosis. These findings were confirmed in vivo, using mice that either globally lacked Hsp70 or overexpressed Hsp70 within the intestinal epithelium. TLR4 activation itself significantly increased Hsp70 expression in enterocytes, which provided a mechanism of autoinhibition of TLR4 signaling in enterocytes. In seeking to define the mechanisms involved, intracellular Hsp70-mediated inhibition of TLR4 signaling required both its substrate-binding EEVD domain and association with the cochaperone CHIP, resulting in ubiquitination and proteasomal degradation of TLR4. The expression of Hsp70 in the intestinal epithelium was significantly decreased in murine and human NEC compared with healthy controls, suggesting that loss of Hsp70 protection from TLR4 could lead to NEC. In support of this, intestinal Hsp70 overexpression in mice and pharmacologic upregulation of Hsp70 reversed TLR4-induced cytokines and enterocyte apoptosis, as well as prevented and treated experimental NEC. Thus, a novel TLR4 regulatory pathway exists within the newborn gut involving Hsp70 that may be pharmacologically activated to limit NEC severity.

Original languageEnglish (US)
Pages (from-to)4543-4557
Number of pages15
JournalJournal of Immunology
Volume188
Issue number9
DOIs
StatePublished - May 1 2012
Externally publishedYes

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HSP70 Heat-Shock Proteins
Intestinal Mucosa
Necrotizing Enterocolitis
Newborn Infant
Enterocytes
Apoptosis
Cytokines
Ubiquitination
Heat-Shock Proteins
Premature Infants
Up-Regulation

ASJC Scopus subject areas

  • Immunology

Cite this

Intracellular heat shock protein-70 negatively regulates TLR4 signaling in the newborn intestinal epithelium. / Afrazi, Amin; Sodhi, Chhinder; Good, Misty; Jia, Hongpeng; Siggers, Richard; Yazji, Ibrahim; Ma, Congrong; Neal, Matthew D.; Prindle, Thomas; Grant, Zachary S.; Branca, Maria F.; Ozolek, John; Chang, Eugene B.; Hackam, David.

In: Journal of Immunology, Vol. 188, No. 9, 01.05.2012, p. 4543-4557.

Research output: Contribution to journalArticle

Afrazi, A, Sodhi, C, Good, M, Jia, H, Siggers, R, Yazji, I, Ma, C, Neal, MD, Prindle, T, Grant, ZS, Branca, MF, Ozolek, J, Chang, EB & Hackam, D 2012, 'Intracellular heat shock protein-70 negatively regulates TLR4 signaling in the newborn intestinal epithelium', Journal of Immunology, vol. 188, no. 9, pp. 4543-4557. https://doi.org/10.4049/jimmunol.1103114
Afrazi, Amin ; Sodhi, Chhinder ; Good, Misty ; Jia, Hongpeng ; Siggers, Richard ; Yazji, Ibrahim ; Ma, Congrong ; Neal, Matthew D. ; Prindle, Thomas ; Grant, Zachary S. ; Branca, Maria F. ; Ozolek, John ; Chang, Eugene B. ; Hackam, David. / Intracellular heat shock protein-70 negatively regulates TLR4 signaling in the newborn intestinal epithelium. In: Journal of Immunology. 2012 ; Vol. 188, No. 9. pp. 4543-4557.
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AU - Afrazi, Amin

AU - Sodhi, Chhinder

AU - Good, Misty

AU - Jia, Hongpeng

AU - Siggers, Richard

AU - Yazji, Ibrahim

AU - Ma, Congrong

AU - Neal, Matthew D.

AU - Prindle, Thomas

AU - Grant, Zachary S.

AU - Branca, Maria F.

AU - Ozolek, John

AU - Chang, Eugene B.

AU - Hackam, David

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N2 - Necrotizing enterocolitis (NEC) is the leading cause of gastrointestinal-related mortality in premature infants, and it develops under conditions of exaggerated TLR4 signaling in the newborn intestinal epithelium. Because NEC does not develop spontaneously, despite the presence of seemingly tonic stimulation of intestinal TLR4, we hypothesized that mechanisms must exist to constrain TLR4 signaling that become diminished during NEC pathogenesis and focused on the intracellular stress response protein and chaperone heat shock protein-70 (Hsp70). We demonstrate that the induction of intracellular Hsp70 in enterocytes dramatically reduced TLR4 signaling, as assessed by LPS-induced NF-κB translocation, cytokine expression, and apoptosis. These findings were confirmed in vivo, using mice that either globally lacked Hsp70 or overexpressed Hsp70 within the intestinal epithelium. TLR4 activation itself significantly increased Hsp70 expression in enterocytes, which provided a mechanism of autoinhibition of TLR4 signaling in enterocytes. In seeking to define the mechanisms involved, intracellular Hsp70-mediated inhibition of TLR4 signaling required both its substrate-binding EEVD domain and association with the cochaperone CHIP, resulting in ubiquitination and proteasomal degradation of TLR4. The expression of Hsp70 in the intestinal epithelium was significantly decreased in murine and human NEC compared with healthy controls, suggesting that loss of Hsp70 protection from TLR4 could lead to NEC. In support of this, intestinal Hsp70 overexpression in mice and pharmacologic upregulation of Hsp70 reversed TLR4-induced cytokines and enterocyte apoptosis, as well as prevented and treated experimental NEC. Thus, a novel TLR4 regulatory pathway exists within the newborn gut involving Hsp70 that may be pharmacologically activated to limit NEC severity.

AB - Necrotizing enterocolitis (NEC) is the leading cause of gastrointestinal-related mortality in premature infants, and it develops under conditions of exaggerated TLR4 signaling in the newborn intestinal epithelium. Because NEC does not develop spontaneously, despite the presence of seemingly tonic stimulation of intestinal TLR4, we hypothesized that mechanisms must exist to constrain TLR4 signaling that become diminished during NEC pathogenesis and focused on the intracellular stress response protein and chaperone heat shock protein-70 (Hsp70). We demonstrate that the induction of intracellular Hsp70 in enterocytes dramatically reduced TLR4 signaling, as assessed by LPS-induced NF-κB translocation, cytokine expression, and apoptosis. These findings were confirmed in vivo, using mice that either globally lacked Hsp70 or overexpressed Hsp70 within the intestinal epithelium. TLR4 activation itself significantly increased Hsp70 expression in enterocytes, which provided a mechanism of autoinhibition of TLR4 signaling in enterocytes. In seeking to define the mechanisms involved, intracellular Hsp70-mediated inhibition of TLR4 signaling required both its substrate-binding EEVD domain and association with the cochaperone CHIP, resulting in ubiquitination and proteasomal degradation of TLR4. The expression of Hsp70 in the intestinal epithelium was significantly decreased in murine and human NEC compared with healthy controls, suggesting that loss of Hsp70 protection from TLR4 could lead to NEC. In support of this, intestinal Hsp70 overexpression in mice and pharmacologic upregulation of Hsp70 reversed TLR4-induced cytokines and enterocyte apoptosis, as well as prevented and treated experimental NEC. Thus, a novel TLR4 regulatory pathway exists within the newborn gut involving Hsp70 that may be pharmacologically activated to limit NEC severity.

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