Intrapulmonary administration of purified NEIL2 abrogates NF-κB–mediated inflammation
2021; Elsevier BV; Volume: 296; Linguagem: Inglês
10.1016/j.jbc.2021.100723
ISSN1083-351X
AutoresNisha Tapryal, Shandy Shahabi, Anirban Chakraborty, Koa Hosoki, Maki Wakamiya, Gobinda Sarkar, Gulshan Sharma, Victor J. Cardenas, István Boldogh, Sanjiv Sur, Gourisankar Ghosh, Tapas K. Hazra,
Tópico(s)Immune Response and Inflammation
ResumoAberrant or constitutive activation of nuclear factor kappa B (NF-κB) contributes to various human inflammatory diseases and malignancies via the upregulation of genes involved in cell proliferation, survival, angiogenesis, inflammation, and metastasis. Thus, inhibition of NF-κB signaling has potential for therapeutic applications in cancer and inflammatory diseases. We reported previously that Nei-like DNA glycosylase 2 (NEIL2), a mammalian DNA glycosylase, is involved in the preferential repair of oxidized DNA bases from the transcriptionally active sequences via the transcription-coupled base excision repair pathway. We have further shown that Neil2-null mice are highly sensitive to tumor necrosis factor α (TNFα)- and lipopolysaccharide-induced inflammation. Both TNFα and lipopolysaccharide are potent activators of NF-κB. However, the underlying mechanism of NEIL2's role in the NF-κB–mediated inflammation remains elusive. Here, we have documented a noncanonical function of NEIL2 and demonstrated that the expression of genes, such as Cxcl1, Cxcl2, Cxcl10, Il6, and Tnfα, involved in inflammation and immune cell migration was significantly higher in both mock- and TNFα-treated Neil2-null mice compared with that in the WT mice. NEIL2 blocks NF-κB's binding to target gene promoters by directly interacting with the Rel homology region of RelA and represses proinflammatory gene expression as determined by co-immunoprecipitation, chromatin immunoprecipitation, and electrophoretic mobility-shift assays. Remarkably, intrapulmonary administration of purified NEIL2 via a noninvasive nasal route significantly abrogated binding of NF-κB to cognate DNA, leading to decreased expression of proinflammatory genes and neutrophil recruitment in Neil2-null as well as WT mouse lungs. Our findings thus highlight the potential of NEIL2 as a biologic for inflammation-associated human diseases. Aberrant or constitutive activation of nuclear factor kappa B (NF-κB) contributes to various human inflammatory diseases and malignancies via the upregulation of genes involved in cell proliferation, survival, angiogenesis, inflammation, and metastasis. Thus, inhibition of NF-κB signaling has potential for therapeutic applications in cancer and inflammatory diseases. We reported previously that Nei-like DNA glycosylase 2 (NEIL2), a mammalian DNA glycosylase, is involved in the preferential repair of oxidized DNA bases from the transcriptionally active sequences via the transcription-coupled base excision repair pathway. We have further shown that Neil2-null mice are highly sensitive to tumor necrosis factor α (TNFα)- and lipopolysaccharide-induced inflammation. Both TNFα and lipopolysaccharide are potent activators of NF-κB. However, the underlying mechanism of NEIL2's role in the NF-κB–mediated inflammation remains elusive. Here, we have documented a noncanonical function of NEIL2 and demonstrated that the expression of genes, such as Cxcl1, Cxcl2, Cxcl10, Il6, and Tnfα, involved in inflammation and immune cell migration was significantly higher in both mock- and TNFα-treated Neil2-null mice compared with that in the WT mice. NEIL2 blocks NF-κB's binding to target gene promoters by directly interacting with the Rel homology region of RelA and represses proinflammatory gene expression as determined by co-immunoprecipitation, chromatin immunoprecipitation, and electrophoretic mobility-shift assays. Remarkably, intrapulmonary administration of purified NEIL2 via a noninvasive nasal route significantly abrogated binding of NF-κB to cognate DNA, leading to decreased expression of proinflammatory genes and neutrophil recruitment in Neil2-null as well as WT mouse lungs. Our findings thus highlight the potential of NEIL2 as a biologic for inflammation-associated human diseases. 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No cancer predisposition or increased spontaneous mutation frequencies in NEIL DNA glycosylases-deficient mice.Sci. Rep. 2017; 7: 4384Crossref PubMed Scopus (17) Google Scholar), their susceptibility to inflammatory stimuli prompted us to explore the mechanistic basis of NEIL2's role in innate immunity. Here, we report that NEIL2 suppresses NF-κB–mediated proinflammatory gene expression. We also demonstrate that NEIL2 interacts with Rel homology region of RelA and blocks NF-κB's promoter occupancy. Importantly, we show that intranasal delivery of endotoxin free recombinant NEIL2 suppresses the NF-κB–mediated immune response in mouse lung. Thus, NEIL2-mediated inhibition of NF-κB signaling may provide a beneficial immunotherapeutic strategy for the prevention of chronic inflammatory human diseases. In an effort to understand the role of NEIL2 in modulating inflammatory gene expression, we induced systemic inflammation in Neil2-null (Neil2−/−) and WT (Neil2+/+) mice via intraperitoneal administration of a single dose of TNFα or phosphate buffered saline (PBS) as mock and conducted a multiplex Real-Time qPCR (RT-qPCR) analysis of 79 inflammation-associated genes (Fig. 1A). Mock- or TNFα-treated Neil2−/− mice lungs presented over 1.5-fold increased expression of ~20 genes (p < 0.05) compared with Neil2+/+ mice (Fig. 1B). Compared with mock-treated Neil2+/+, TNFα treatment increased expression of 25 inflammatory genes over 2-fold (p < 0.05) in Neil2+/+ mice (Fig. 1C, left); however, following TNFα treatment, 48 genes were upregulated more than a 2-fold (p < 0.05) in Neil2−/− mice (Fig. 1C, right). TNFα-treated Neil2−/− lungs presented increased expression of genes involved in neutrophil trafficking (Cxcl1and Cxcl2), the Th1 response, natural killer cells, or monocyte trafficking (Cxcl10, Ccl3, and ccl2), as well as in the regulation of immune response, hematopoiesis, or inflammation (Tnfα, Il6, and IL1β) causing inflammation of lungs. The multiplex gene array data were further validated using RT-qPCR (list of primers; Table S1) in lungs (Fig. 1D) and in two additional tissues, brain and muscle (Fig. S1, A and B) with similar results, indicating a systemic inflammatory response. Collectively, these data imply that NEIL2 deficiency alone results in a higher basal level of systemic inflammation that can be aggravated further on exposure to an external inflammatory stimulus. Under pathophysiological conditions, neutrophils are recruited to the lungs in response to chemokines, expression of which are induced because of infections and/or physical or chemical injuries (37Williams A.E. Chambers R.C. The mercurial nature of neutrophils: Still an enigma in ARDS?.Am. J. Physiol. Lung Cell Mol. Physiol. 2014; 306: L217-230Crossref PubMed Scopus (228) Google Scholar). Therefore, to better understand the physiological role of NEIL2 in modulating lung inflammation, we delivered TNFα or PBS (as mock) to Neil2+/+ or Neil2−/− mice via intranasal (i.n.) route and analyzed the expression of proinflammatory genes in the lungs over a period of 120 min by RT-qPCR. The basal level of Cxcl1, Cxcl2, Cxcl10, Il6, Tnfα, and ILβ expression in Neil2−/− mice lung was significantly higher compared with Neil2+/+ (Fig. 2A). TNFα treatment resulted in further increase in expression of all the genes tested, especially in Neil2−/− compared with Neil2+/+ mice lungs (Figs. 2B and S2). CXC chemokines are strong chemoattractants, and their enhanced expression plays a critical role in modulating neutrophil and lymphocyte migration (38Benigni G. Dimitrova P. Antonangeli F. Sanseviero E. Milanova V. Blom A. van Lent P. Morrone S. Santoni A. Bernardini G. CXCR3/CXCL10 Axis regulates neutrophil-NK cell cross-talk determining the severity of experimental osteoarthritis.J. Immunol. 2017; 198: 2115-2124Crossref PubMed Scopus (31) Google Scholar, 39Rajarathnam K. Schnoor M. Richardson R.M. Rajagopal S. How do chemokines navigate neutrophils to the target site: Dissecting the structural mechanisms and signaling pathways.Cell Signal. 2019; 54: 69-80Crossref PubMed Scopus (51) Google Scholar). Neutrophil and lymphocyte levels in bronchoalveolar lavage fluid (BALF) of mock-treated mice were low (Fig. 2C, far left panel). However, following TNFα treatment, Neil2−/− mice showed significantly higher neutrophil (~3-fold; p = 0.0052) (Fig. 2C, middle and far right panels, and Fig. 2D) recruitment in the BALF compared with Neil2+/+ mice. These data are consistent with our earlier report (17Chakraborty A. Wakamiya M. Venkova-Canova T. Pandita R.K. Aguilera-Aguirre L. Sarker A.H. Singh D.K. Hosoki K. Wood T.G. Sharma G. Cardenas V. Sarkar P.S. Sur S. Pandita T.K. Boldogh I. et al.Neil2-null mice accumulate oxidized DNA bases in the transcriptionally active sequences of the genome and are susceptible to innate inflammation.J. Biol. Chem. 2015; 290: 24636-24648Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Additionally, we also observed a significantly higher number of lymphocytes (~4-fold; p = 0.02) in TNFα-treated lungs (Fig. 2E). These data are in accordance with the results observed with systemic inflammation in Neil2−/− versus Neil2+/+ mice (Fig. 1) and further confirm that NEIL2-deficient mice not only has higher basal inflammatory state but also experience a greater insult from proinflammatory mediators following exposure to inflammatory agents. NF-κB is directly involved in the transcriptional regulation of a variety of chemokines/cytokines (40Liu T. Zhang L. Joo D. Sun S. NF-κB signaling in inflammation.Signal. Transduct. 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Intriguingly, a peak NEIL2 occupancy was also detected at the promoter sites of all the genes tested in mock-treated groups (Fig. 3B, 0 min); however, such binding of NEIL2 decreased significantly over time following TNFα treatment with the concurrent increase in NF-κB occupancy at the same loci (Fig. 3B versus A). The inverse nature of NEIL2 and NF-κB occupancy at proinflammatory gene promoters suggests an interplay between NEIL2 and NF-κB at promoter sites and may explain the NEIL2-mediated modulation of inflammatory genes. To test whether NEIL2 modulates NF-κB's binding to its cognate motif, we analyzed NF-κB binding by electrophoretic mobility-shift assay (EMSA) using the nuclear extracts (NEs) from PBS/TNFα-treated Neil2+/+ or Neil2−/− mice lungs with radiolabeled oligonucleotides (oligos) containing the NF-κB DNA response element or κB-motif present in the promoters of Cxcl1, Cxcl2, and Il6. Neil2−/− mice displayed a significantly higher level of NF-κB-DNA complex in both mock (-) and TNFα-treated (+) groups with the WT-Cxcl1 oligos (Fig. 3C, lane 3 versus 1 and lane 4 versus 2, respectively) and -Cxcl2 or -Il6 oligos (Fig. S3, A and C, lane 3 versus 1 and lane 4 versus 2, respectively). The NF-κB–DNA complex was competed out effectively by a 100-fold molar excess of unlabeled C1-WT, C2-WT, and IL6-WT, but not by a mutant -κB oligo C1-M, C2-M, and IL6-M (Fig. 3C, lane 8 versus 7, Fig. S3B, lane 3 versus 2 and Fig. S3C, lane 8 versus 7, respectively). The mutant oligos lacked binding with recombinant RelA (Fig. S3D), confirming the specificity of the binding with WT κB-motif. The addition of anti-RelA antibody to the NEs caused a shift of the protein–DNA complex band with both C1-WT and C2-WT oligos, confirming the identity of RelA in the complex (Fig. 3D). Furthermore, siRNA-mediated NEIL2 depletion in mouse lung epithelial (MLE12) cells (Fig. S3E) also resulted in higher NF-κB–DNA binding compared with NEIL2-proficient cells, with or without TNFα-treatment (Fig. 3E, lane 3 versus 1 and lane 4 versus 2). All these data strongly suggest that NEIL2 modulates NF-κB's promoter binding activity and subsequent gene expression, and provides an opportunity to therapeutically modulate the detrimental effects of inflammation. To investigate the mechanism by which NEIL2 acts as a negative modulator of NF-κB, we first tested whether overexpression of NEIL2 directly inhibits NF-κB's DNA binding. Ectopically expressed C-terminal FLAG-tagged NEIL2 (NEIL2-FLAG) in human lung 358 (h358) cells where NF-κB is constitutively active and is present in the nucleus (43Yang L. Zhou Y. Li Y. Zhou J. Wu Y. Cui Y. Yang G. Hong Y. Mutations of p53 and KRAS activate NF-κB to promote chemoresistance and tumorigenesis via dysregulation of cell cycle and suppression of apoptosis in lung cancer cells.Cancer Lett. 2015; 357: 520-526Crossref PubMed Scopus (87) Google Scholar) or MLE12 cells (Fig. S4, A and B) significantly decreased NF-κB-DNA complex formation at κB-site in both the cell lines compared with cells transfected with empty-vector (Fig. 4A, lane 3 versus 2 and Fig. 4B, lane 3 versus 2 and 5 versus 4). Next, we performed EMSA with the NEs prepared from TNFα-treated mouse lungs and found that addition of cell purified NEIL2 significantly blocked formation of the NF-κB-DNA complex in a dose dependent manner (Fig. 4C, lanes 2–4 versus 1). To test whether NEIL2-mediated inhibition of the NF-κB-motif binding is due to protein–protein interaction, we immunoprecipitated NEIL2 using the anti-FLAG antibody from the human bronchial epithelial cell line BEAS-2B stably expressing NEIL2-FLAG and detected RelA in the immunocomplex as early as 15 min following TNFα treatment (Fig. 4D). Purified NEIL2-FLAG from h358 cells also pulled down recombinant RelA (Fig. 4E). Furthermore, purified NEIL2-FLAG specifically co-immunoprecipitated the DNA-binding Rel homology region (RHR) of RelA (GST-RHR), but not the transcription-activation domain of RelA (GST-TAD) (Fig. 4F), suggesting that NEIL2 inhibits the RelA:DNA complex formation by interacting with DNA-binding domain of RelA. Thus, it is conceivable that NEIL2's association with the DNA binding domain of RelA prevents RelA from binding to the κB site and consequently suppresses the proinflammatory gene expression. Given the antiinflammatory properties of NEIL2, we attempted to evaluate its potential as a therapeutic biologic for inflammatory diseases. We delivered an optimized amount of endotoxin-free His-tagged recombinant NEIL2 (rNEIL2, E.coli purified) via the intranasal route to the lungs of age- and sex-matched Neil2+/+ and Neil2−/− mice using a synthetic peptide, K16SP, as a carrier (44Mahlum E. Mandal D. Halder C. Maran A. Yaszemski M.J. Jenkins R.B. Bolander M.E. Sarkar G. Engineering a noncarrier to a highly efficient carrier peptide for noncovalently delivering biologically active proteins into human cells.Anal. Biochem. 2007; 365: 215-221Crossref PubMed Scopus (9) Google Scholar). Mice were then treated with TNFα, 72 h post-NEIL2 treatment, and lungs were collected for differential cell count, RT-qPCR, and NE preparations. The enzymatically inactive heat-inactivated rNEIL2 (HI-rNEIL2) (Fig. S5A) was used as a control. HI-rNEIL2 and active rNEIL2 (Act-rNEIL2) were uniformly delivered to male/female mouse lungs (Fig. S5, B and C). Act-rNEIL2 blocked neutrophil count in BALF, 16 h post mock (Fig. S5D) and TNFα treatment (Fig. 5, A and B) in both Neil2+/+ and Neil2−/− male or female mice compared with HI-rNEIL2 transduced groups. Similarly, Act-rNEIL2 decreased the recruitment of TNFα-induced lymphocytes (Fig. 5C) in both Neil2+/+ and Neil2−/− groups. Chemokine/cytokine expression was significantly suppressed in Act-rNEIL2 recipient mouse lungs, 1 h post mock or TNFα treatment as analyzed by RT-qPCR (Fig. 5D). Furthermore, a remarkable decrease in NF-κB-DNA complex formation was observed in Act-rNEIL2 transduced Neil2+/+ and Neil2−/− male mice lung NEs compared with HI-rNEIL2-transduced groups, as analyzed by EMSA using Cxcl1, Cxcl2, Il6, or Tnfα oligo probes (Figs. 5E and S6, lanes 2, 4, 7 and 9 versus 1, 3, 6 and 8, respectively, and Fig. 5F). Similar results were observed with the NEs prepared from Act-NEIL2 transduced Neil2+/+ and Neil2−/− female mice lungs for all tested probes (Fig. S7). Collectively, these data provide strong evidence for the therapeutic potential of NEIL2 to actively block NF-κB–mediated inflammatory responses by directly suppressing NF-κB's binding to its consensus motifs within the target gene promoters. Persistent activation of inflammatory molecules plays a central role in many chronic human diseases. Atypical NF-κB signaling underlies the uncoordinated expression of prototypical proinflammatory cytokines which promotes the pathogenesis of inflammatory dis
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