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Mechanisms by which two different Toll-like receptor ligands induce divergent immune responses in the lungs


The mammalian immune system can mount several different types of innate and adaptive responses. The choice of the type of immune response depends on a variety of factors, including which innate immune receptors recognize conserved pathogen molecules on the invading pathogen. How allergens and other type 2 stimuli, such as parasitic worm infections, induce type 2 immunity have been intensely studied, but it is still unclear which pathways are critical for driving this type of immune response. Interestingly, although Toll-like receptor (TLR) stimulation often induces type 1 immune responses during bacterial or viral infections, it can also promote type 2 immune responses in the lung in some circumstances, and this may be important for development of asthma. The work presented in this dissertation investigate the mechanisms by which two different TLR ligands induce divergent immune responses in the lungs.

In Chapter 3, I describe experiments using an intranasal (i.n.) sensitization and challenge model to characterize and understand how a synthetic TLR9 ligand, unmethylated cytosine followed by guanosine (CpG) oligodeoxyribonucleotide (ODN), which has great promise of being a potent adjuvant in vaccines and an immunomodulator clinically, induces an immune response in the lungs. I demonstrated that the sensitization i.n. with the model antigen ovalbumin (OVA) plus CpG ODN as adjuvant, and subsequent rechallenge with OVA led to a Th1 response in the lungs and increased serum OVA-specific IgG2c. Early after CpG ODN and OVA i.n. administration, increased levels mRNA encoding IL-12 p40, and increased numbers of dendritic cells (DCs) and monocytes were present in the lungs. CpG ODN treatment led to the elevation of costimulatory molecules on DCs and monocytes in the lungs, and on the migratory DCs in the draining mediastinal lymph node (LN). I.n. CpG ODN was also able to induce early IFN-gamma production in both innate-like lymphocytes such as gamma delta T cells, natural killer (NK) T cells, and NK cells, and also in adaptive CD4 T cells and CD8 T cells. Strikingly, these innate and adaptive immune responses were dependent on myeloid differentiation primary response 88 (MyD88) signaling in DCs, and most production of IFN-gamma was dependent on IL-12, which was produced during the innate phase of the response. Based on these results, we suggest that DCs directly sense CpG ODN in the lungs via TLR9 and that this recognition both induces their maturation and their production of IL-12, which promotes innate production of IFN-gamma and the Th1 response.

Chapter 4 describes studies in which CpG ODN and the TLR5 and TLR11 ligand, flagellin, were compared to examine how TLRs can promote different adaptive immune responses in the lungs. In contrast to mice i.n. sensitized with CpG ODN plus OVA, those sensitized with flagellin plus OVA exhibited an innate inflammatory response dominated by neutrophils and monocytes, developed a predominant Th2 response to OVA, and made substantial amounts of IgE anti-OVA antibodies. Antigenic re-challenge of the mice sensitized with flagellin plus OVA, but not the mice sensitized with CpG ODN plus OVA, led to a vigorous lung inflammation dominated by eosinophils, as expected for a Th2 response. Strikingly, the early cytokine responses in the lung to the two different TLR ligands showed a number of important differences. CpG ODN induced much higher levels of mRNA encoding IL-12 p40, whereas, flagellin preferentially induced TSLP mRNA and secretion of the mature form of IL-33, and also induced elevated levels of mRNAs encoding IL-1-alpha and IL-1-beta. In both cases, migratory DCs in the draining LN after stimulation with ligands for either TLR5/TLR11 or TLR9 had strong induction of the costimulators CD80 and CD86, but only CpG ODN also induced upregulation of CD40. Interestingly, MyD88 signaling in DCs was partially required for the flagellin-induced upregulation of CD80 on migratory DCs, and for the IL-4 production by CD4 T cells in the draining LN on d6. These results indicate that although TLR5, TLR11, and TLR9 all signal via the signaling adaptor MyD88, the innate cytokine response to these TLRs is quite different, and the distinctive innate cytokine production results in distinctive polarization of the adaptive humoral and cell-mediated immune responses.

These studies have provided insight into how TLR stimulation can drive Th1 and Th2 responses in the lungs, and how complex immune responses occur in the lungs, which have important implications for understanding infectious diseases and asthma, and for vaccine design.

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