Search

Supplemental material, Additional_file_2_revised_13-06-2018 for Association of DFNA5, SYK, and NELL1 variants along with HPV infection in oral cancer among the prolonged tobacco-chewers by Sharbadeb Kundu, Vijayalakshmi Ramshankar, Akalesh Kumar Verma, Soundara Viveka Thangaraj, Arvind Krishnamurthy, Rajeev Kumar, Ravi Kannan and Sankar Kumar Ghosh in Tumor Biology

Supplemental material, Addional_file_1_revised_13-06-2018 for Association of DFNA5, SYK, and NELL1 variants along with HPV infection in oral cancer among the prolonged tobacco-chewers by Sharbadeb Kundu, Vijayalakshmi Ramshankar, Akalesh Kumar Verma, Soundara Viveka Thangaraj, Arvind Krishnamurthy, Rajeev Kumar, Ravi Kannan and Sankar Kumar Ghosh in Tumor Biology

Supplemental material, Supplementary_Information_revised_13-06-2018 for Association of DFNA5, SYK, and NELL1 variants along with HPV infection in oral cancer among the prolonged tobacco-chewers by Sharbadeb Kundu, Vijayalakshmi Ramshankar, Akalesh Kumar Verma, Soundara Viveka Thangaraj, Arvind Krishnamurthy, Rajeev Kumar, Ravi Kannan and Sankar Kumar Ghosh in Tumor Biology

The activation of pro-inflammatory gene programs by nuclear factor-kappaB (NF-kappaB) is primarily regulated through cytoplasmic sequestration of NF-kappaB by the inhibitor of kappaB (IkappaB) family of proteins. IkappaBbeta, a major isoform of IkappaB, can sequester NF-kappaB in the cytoplasm, although its biological role remains unclear. Although cells lacking IkappaBbeta have been reported, in vivo studies have been limited and suggested redundancy between IkappaBalpha and IkappaBbeta. Like IkappaBalpha, IkappaBbeta is also inducibly degraded; however, upon stimulation by lipopolysaccharide (LPS), it is degraded slowly and re-synthesized as a hypophosphorylated form that can be detected in the nucleus. The crystal structure of IkappaBbeta bound to p65 suggested this complex might bind DNA. In vitro, hypophosphorylated IkappaBbeta can bind DNA with p65 and c-Rel, and the DNA-bound NF-kappaB:IkappaBbeta complexes are resistant to IkappaBalpha, suggesting hypophosphorylated, nuclear IkappaBbeta may prolong the expression of certain genes. Here we report that in vivo IkappaBbeta serves both to inhibit and facilitate the inflammatory response. IkappaBbeta degradation releases NF-kappaB dimers which upregulate pro-inflammatory target genes such as tumour necrosis factor-alpha (TNF-alpha). Surprisingly, absence of IkappaBbeta results in a dramatic reduction of TNF-alpha in response to LPS even though activation of NF-kappaB is normal. The inhibition of TNF-alpha messenger RNA (mRNA) expression correlates with the absence of nuclear, hypophosphorylated-IkappaBbeta bound to p65:c-Rel heterodimers at a specific kappaB site on the TNF-alpha promoter. Therefore IkappaBbeta acts through p65:c-Rel dimers to maintain prolonged expression of TNF-alpha. As a result, IkappaBbeta(-/-) mice are resistant to LPS-induced septic shock and collagen-induced arthritis. Blocking IkappaBbeta might be a promising new strategy for selectively inhibiting the chronic phase of TNF-alpha production during the inflammatory response.

Toll-like receptors (TLRs) are innate immune receptors that sense a variety of pathogen-associated molecular patterns (PAMPs) by interacting with them and subsequently initiating signal transduction cascades that elicit immune responses. TLR11 has been shown to interact with two known protein PAMPs: Salmonella and E. coli flagellin FliC and Toxoplasma gondii profilin-like protein. Given the highly divergent biology of these pathogens recognized by TLR11, it is unclear whether common mechanisms are used to recognize these distinct protein PAMPs. Here we show that TLR11 interacts with these two PAMPs using different receptor domains. Furthermore, TLR11 binding to flagellin and profilin exhibits differential dependency on pH and receptor ectodomain cleavage.

Excessive type 2 helper T cell responses to environmental antigens can cause immunopathology such as asthma and allergy, but how such immune responses are induced remains unclear. We studied this process in the airways by immunizing mice intranasally with the antigen ovalbumin together with either of two Toll-like receptor (TLR) ligands. We found the TLR5 ligand flagellin promoted a type 2 helper T cell response, whereas, a TLR9 ligand CpG oligodeoxyribonucleotide (ODN) promoted a type 1 helper T cell response. CpG ODN induced mRNA encoding interleukin (IL)-12 p40, whereas, flagellin caused IL-33 secretion and induced mRNAs encoding IL-1 and thymic stromal lymphopoietin (TSLP). By using mice deficient in the TLR and IL-1R signaling molecule, myeloid differentiation primary response 88 (MyD88), in conventional dendritic cells (cDCs) and alveolar macrophages (AMs), and by cell sorting different lung populations after 2 hours of in vivo stimulation, we characterized the cell types that rapidly produced inflammatory cytokines in response to TLR stimulation. CpG ODN was likely recognized by TLR9 on cDCs and AMs, which made mRNA encoding IL-12. IL-12 was necessary for the subsequent innate and adaptive interferon-γ production. In contrast, flagellin stimulated multiple cells of hematopoietic and non-hematopoietic origin, including AMs, DCs, monocytes, and lung epithelial cells. AMs were largely responsible for IL-1α, whereas lung epithelial cells made TSLP. Multiple hematopoietic cells, including AMs, DCs, and monocytes contributed to other cytokines, including IL-1β and TNFα. MyD88-dependent signals, likely through IL-1R and IL-33R, and MyD88-independent signals, likely from TSLP, were necessary in cDCs for promotion of the early IL-4 response by CD4 T cells in the draining lymph node. Thus, the cell types that responded to TLR ligands were a critical determinant of the innate cytokines produced and the character of the resulting adaptive immune response in the airways.