<p> Carrageenan, a mixture of sulphated galactans extractable by hot water from certain marine algae, has been known for its activity in a reversible stimulation of connective tissue growth. One subcutaneous injection of this polysaccharide elicits a granuloma formation which regresses after a few weeks. A single injection after regression results in heightened response; this suggested an immunological reaction. Serial injections of either lambda or kappa carrageenan into rabbits resulted in the formation of precipitins. The present study gives both qualitative and quantitative studies of the reaction involved. Fractions of carrageenans from different species of marine algae were tested for their ability to precipitate either anti-lambda carrageenan antiserum or anti-kappa carrageenan antiserum. Deductions were made from the data as to the structure of the polysaccharides.</p> / Thesis / Master of Science (MSc)

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Long non-coding RNAs (lncRNAs) have been implicated as important regulators of inflammation through various mechanisms in both the innate and adaptive immune systems of mice and humans. The majority of SNPs identified by GWAS to be associated with autoimmune disorders lie within non-coding areas of the genome, including genes for lncRNAs. To identify lncRNAs with relevancy to inflammation and autoimmunity, a discovery pipeline was used to find lncRNAs differentially expressed in TLR4 activated murine macrophages, conserved between mice and humans, and harboring GWAS identified SNPs associated with autoimmune disorders. Two of the six candidate lncRNAs identified, Lnc15 and Lnc13, are decreased in activated macrophages and are associated with both celiac and Crohn’s disease. To further explore the regulation and influence of these two lncRNAs during inflammation and its resolution, a variety of in vitro and in vivo techniques were utilized, including novel mouse knockout models. An investigation of Lnc15 was conducted in cells of both the innate and adaptive immune system, where the dominant isoform of Lnc15 was identified to be a ~1.4 kb transcript localized to the cytoplasm in both murine macrophages and T cells. Analysis of Lnc15 regulation was conducted in activated murine macrophages, focused on TLR signaling. Through stimulating macrophages with specific TLR ligands, Lnc15 was found to be decreased by TLR2, TLR3, and TLR4 signaling, likely dependent upon both MYD88 and TRIF. While not dependent upon NF-κB, protein synthesis is required for TLR induced decreases in Lnc15 levels. Conversely, activated neutrophils significantly increase Lnc15 levels, although the mechanism of regulation is not yet known. Mice lacking Lnc15 globally were found to be more susceptible to DSS induced colitis, which is likely dependent upon a defect in the innate immune system. In the adaptive immune system, Lnc15 was found to be specifically upregulated in Tregs compared to other T cell subsets. Lnc15 deficient Tregs had a reduced suppressive capacity in vitro, but not in vivo in a T cell induced model of colitis. These findings suggest Lnc15 plays a role in Treg suppressive capacity under certain conditions, but the exact mechanism influenced remains to be identified. Additionally, overexpression of Lnc15 in a murine T cell line resulted in a decrease in Rorc expression. A Lnc15 RNA pulldown experiment identified USF2, a transcription factor known to regulate Rorc expression, and UBR5, a ubiquitin-protein ligase known to influence RORyt stability, as protein interactors of Lnc15. These data indicate that Lnc15 can influence aspects of RORyt biology, which implicates Lnc15 as a regulator of either the plasticity between Tregs and Th17 cells, or Treg ability to suppress inflammatory Th17 cells. An investigation into Lnc13 regulation by disease relevant cytokines was conducted with a series of macrophage stimulation experiments. Lnc13 was found to be positively regulated by cytokines with an anti-inflammatory capacity, including IL-6, IL-4 and IL-10. When Lnc13 deficient macrophages were polarized, a higher expression of Il6 was detected in both M1 and M2 macrophages, suggesting a regulatory connection between Lnc13 and IL-6 across macrophage activation states. Previously identified Lnc13 target genes displayed a quicker transcriptional response to LPS stimulation in Lnc13 deficient macrophages. Additionally, when the Lnc13 mouse was crossed with the DQ8 transgenic mouse model and challenged to gluten, the ileum tissue of Lnc13 deficient mice expressed higher amounts of Il12 and Ifng, cytokines directly relevant to celiac disease. These findings provide support for Lnc13 as a novel regulator of macrophage response and cytokine expression in response to disease relevant stimuli.

During tumor growth there are a series of phenotypic and functional changes that occur in macrophages (M_Φ) that ultimately lead to the immunosuppression of the tumor-bearing host (TBH). To investigate the phenotypic changes of M_Φ during tumor growth, we examined the expression of the M_Φ surface antigens, Mac-1, Mac-2, Mac-3, and Ia on peritoneal and splenic M_Φ. In the peritoneal cavity there was no change in the percentage of Mac-1⁺ M_Φ but a decrease in the percentage of Mac-2⁺, -3⁺, and Ia⁺ M_Φ during tumor growth. In addition, three distinctly sized populations of peritoneal M_Φ, showing differential antigen expression, also shifted during tumor growth. In the peritoneal cavity there was a decrease in the percentage of M_Φ co-expressing the Mac-2, -3, and Ia antigens, leading to a shift towards Mac-1⁺ 2⁻ 3⁻ Ia⁻ TBH M_Φ. In splenic M_Φ, the percentage of Mac-1⁺, -2⁺, and -3⁺ M_Φ increased, while the percentage of Ia⁺ M_Φ decreased. Splenic M_Φ showed an increase in the percentage of M_Φ co-expressing Mac-1, -2, and -3 antigens and a decrease in the percentage of M_Φ co-expressing Ia, leading to a shift towards a Mac-1⁺ 2⁺ 3⁺ Ia⁻ TBH M_Φ. Taken together, these data suggest that tumor growth alters the phenotype of M_Φ and causes a shift in M_Φ subpopulations. After measuring the phenotypic changes in M_Φ during tumor growth, changes in M_Φ accessory function to T cells were assessed. TBH M_Φ have significantly reduced accessory activity for autoreactive T cells. This reduction is caused by decreased Ia antigen expression and increased production of the suppressor molecule, prostaglandin (PG). TBH M_Φ down-regulated autoreactive T cell responsiveness to interleukin (IL)-1, IL-2, and IL-4. In addition to TBH M_Φ reducing T cell responsiveness to cytokines, TBH CD4⁺ T cells alone were less responsive to the cytokines IL-1, IL-2, and IL-4. To examine the responsiveness of M_Φ to activation signals, lipopolysaccharide (LPS) was incubated with normal and TBH splenic M_Φ and assessed for their phenotypic, functional, and cell-cycle changes. The data showed that TBH M_Φ had a decreased responsiveness to LPS. We showed that there was a shift from an Ia⁺ M_Φ in the normal host to an Ia⁻ M_Φ in the TBH. Concomitant with the shift in TBH M_Φ Ia⁻ phenotype was a change in TBH M_Φ function. Normal and TBH Ia⁻ M_Φ were suppressor M_Φ. TBH Ia⁻ M_Φ, however, suppressed autoreactive and alloreactive CD4⁺ T cells significantly more than could their normal counterparts. Tumor growth causes quantitative and qualitative changes in Ia⁻ suppressor M_Φ. Although Ia⁻ M_Φ-mediated suppression seemed to be the major source of down-regulation of CD4⁺ T cells, CD8⁺ T cells were not without fault. In the TBH, there was an increase in the percentage of CD8⁺ T cells and an increase in CD8⁺ T cell-mediated suppression. In conclusion, tumor growth leads to a change in immunoregulation that causes suppression of the immune response. / Ph. D.

Call number: LD2668 .T4 1985 G733 / Master of Science