Tumor suppressor CYLD acts as a negative regulator for non-typeable Haemophilus influenza-induced inflammation in the middle ear and lung of mice

Abstract

Non-typeable Haemophilus influenza (NTHi) is an important human pathogen causing respiratory tract infections in both adults and children. NTHi infections are characterized by inflammation, which is mainly mediated by nuclear transcription factor kappaB (NF-kappaB)-dependent production of inflammatory mediators. The deubiquitinating enzyme cylindromatosis (CYLD), loss of which was originally reported to cause a benign human syndrome called cylindromatosis, has been identified as a key negative regulator for NF-kappaB in vitro. However, little is known about the role of CYLD in bacteria-induced inflammation in vivo. Here, we provided direct evidence for the negative role of CYLD in NTHi-induced inflammation of the mice in vivo. Our data demonstrated that CYLD is induced by NTHi in the middle ear and lung of mice. NTHi-induced CYLD, in turn, negatively regulates NTHi-induced NF-kappaB activation through deubiquitinating TRAF6 and 7 and down-regulates inflammation. Our data thus indicate that CYLD acts as a negative regulator for NF-kappaB-dependent inflammation in vivo, hence protecting the host against detrimental inflammatory response to NTHi infection.

Figure 1. Cyld^−/− mice are hyperresponsive to NTHi-induced inflammation in the ear and lung of the mice.

A, NTHi was trans-tympanically inoculated into the middle ears of WT and Cyld^−/− mice and bullaes were dissected from WT and Cyld^−/− mice inoculated with NTHi and saline for the control for histological analysis (H&E stain, 200X). B, NTHi was intratracheally inoculated into the lungs of WT and Cyld^−/− mice and lung tissues were dissected from WT and Cyld^−/− mice inoculated with NTHi and saline for the control for histological analysis (H&E stain, 200×). C & D, NTHi was trans-tympanically (C) or intratracheally (D) inoculated into the middle ear (C) or lung (D), respectively, of WT and Cyld^−/− mice and mRNA expression of the inflammatory mediators, IL-1β and MIP-2, was measured by Q-PCR analysis *p<0.005 compared with control inoculation in WT mice, **p<0.05 compared with NTHi inoculation in WT mice. Values are means ± S.D. (n = 3). CON, control.

Figure 2. NTHi-induced CYLD is responsible for down-regulation of inflammatory response.

A & B, NTHi induced CYLD expression at both mRNA (A) and protein levels (B) in HMEEC-1 cells. C, NTHi induced CYLD expression at the mRNA level in the middle ear of WT mice. D & E, NTHi induced expression of CYLD and IL-1β (D) or MIP-2 (E) at the mRNA level in the lung of WT mice. *p<0.05 compared with CON. Values are means ± S.D. (n = 3). CON, control.

Figure 3. NTHi induces inflammatory response through TLR2-MyD88-TRAF6/7-NF-κB signaling pathway.

A & B, Overexpressing dominant-negative (DN) mutants of TLR2, MyD88 and TRAF6/7 inhibited NTHi-induced activation of NF-κB (A) and up-regulation of IL-1β and IL-8 (B) in HMEEC-1 cells. *p<0.001, compared with Mock. Values are means ± S.D. (n = 3). C, NTHi-induced expressions of IL-1β and MIP-2 mRNA were reduced in the lung of Tlr2^−/− mice in vivo. *p<0.005, compared with CON in WT mice, **p<0.05 compared with NTHi inoculation in WT mice. Values are means ± S.D. (n = 3). D, NTHi-induced expressions of IL-1β and MIP-2 were reduced in the lung of MyD88^+/− mice in vivo. E, Histological analysis of lung of Tlr2^−/− mice revealed reduced inflammatory response by NTHi compared with WT mice (H&E stain, ×200).

Figure 4. CYLD acts as a negative regulator for NTHi-induced NF-κB activation via negative cross-talk with TRAF6/7.

A. CYLD knockdown by siRNA-CYLD markedly reduced expression of CYLD and enhanced NTHi-induced NF-κB-dependent promoter activity in HMEEC cells. B, overexpression of WT-CYLD inhibited NTHi-induced NF-κB-dependent promoter activity in HMEEC cells. C, NTHi-induced DNA binding activity of NF-κB was enhanced by CYLD knockdown, as assessed by EMSA. D, CYLD knockdown by siRNA-CYLD enhanced NTHi-induced IL-1β and IL-8 expression at mRNA level in HMEEC-1 cells. E, TRAF7 synergistically enhanced TRAF6-induced NF-κB-dependent promoter activity. Overexpression of WT-CYLD inhibited TRAF6- and TRAF7-induced NF-κB-dependent promoter activity, whereas CYLD knockdown by siRNA-CYLD enhanced TRAF6- and TRAF7-induced NF-κB-dependent promoter activity in lung epithelial A549 cells. *p<0.05, compared with NTHi-inoculated in Mock in A, B and D. Values are means ± S.D. (n = 3).

Figure 5. CYLD inhibits TRAF6 and TRAF7 in a deubiquitination-dependent manner.

A, NTHi induced ubiquitination of TRAF6. B, co-expressing WT-CYLD inhibited, whereas CYLD knockdown by siRNA-CYLD enhanced the ubiquitination of TRAF6 in HeLa cells. C, NTHi induced ubiquitination of TRAF7. D, co-expressing WT-CYLD inhibited, whereas CYLD knockdown by siRNA-CYLD enhanced the ubiquitination of TRAF7 in HeLa cells.

Figure 6. Schematic representation of the negative regulation of NTHi-induced inflammatory response by CYLD.