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Throughout life the immune system is faced with the challenging task of appropriately responding to both self- and nonself-derived ligands. The innate immune system utilizes pattern-recognition receptors, such as Toll-like receptors (TLR), to identify specific features of microbes, while the adaptive immune system employs T cells and antibodies (IgA, IgG) to generate robust secondary responses to invasive microorganisms. However, this poses the potential for inappropriate recognition of self-derived ligands or symbiotic bacteria. Therefore, the balance between recognition of non-self while preserving tolerance to self is necessary to prevent autoimmunity and detrimental infections. To better understand this balance, I first examined how the innate immune system prevents recognition of self-nucleic acids. In this study, I explored the consequences of dysregulating compartmentalized activation of a nucleic acid sensing receptor, TLR9. By inducing in vivo expression of a TLR9 mutant (TLR9TM) that bypasses the need for intracellular activation, I discovered that dysregulated TLR9 activation early in life drives a fatal inflammatory disease, driven by type II interferon. In contrast, induced expression of TLR9TM late in life led to a milder, systemic autoinflammatory disease. This study demonstrates that compartmentalized activation of TLR9, especially early in life, is necessary to prevent deleterious recognition of self-nucleic acids. Early in life the naïve immune system must be able to differentiate pathogenic microbes from symbiotic, beneficial microbes. The naïve immune system receives some instruction through acquisition of maternal-derived microbiota-reactive antibodies, such as IgG2b and IgG3. These antibodies are anti-inflammatory and help to suppress inappropriate immune responses to commensal bacteria in the neonatal gut. However, the exact mechanism(s) by which maternal-derived IgG2b and IgG3 instruct the naïve immune system remain relatively unknown. To better understand the role of microbiota-reactive IgGs in tuning the immature immune system, I generated mice that produce pro-inflammatory microbiota-reactive IgG2c (termed IgG32c/2c mice). Examination of the IgG32c/2c mice revealed that acquisition of maternal-derived microbiota-reactive IgG2c leads to fatal disease. This disease was characterized by weight loss and intestinal inflammation as measured by an increase in fecal lipocalin-2, an increase in intestinal myeloperoxidase, and an expansion of CD11b+ myeloid cells in the spleen. In contrast, neonates that acquire maternal-derived microbiota-reactive IgG3 do not experience weight loss, inflammation, or fatality. This study establishes that effector functions elicited by maternal-derived microbiota-reactive antibodies help dictate neonatal fitness by fine-tuning the responses generated by the naïve immune system to symbiotic gut bacteria.The work in this dissertation establishes how the innate immune system regulates nucleic-acid sensing TLRs to prevent inappropriate recognition of self-derived ligands. In addition, the work in this dissertation reveals how maternal-derived antibodies instruct the naïve immune system in how to respond to beneficial microbes early in life.

Systemic lupus erythematosus (SLE) is a highly heterogeneous and prevalent autoimmune disease. The development of anti-nuclear autoantibodies (ANAs) is a hallmark of SLE and many patients will go on to develop glomerulonephritis (GN), a potentially fatal destruction of the kidneys. In the CD45E613R murine model of SLE, a single point mutation in the juxtamembrane wedge region of the protein tyrosine phosphatase CD45, results in hyperactivated immunoreceptor tyrosine-based activation motif (ITAM) signaling. Interestingly, despite the mutation functioning biochemically identically, different strains expressing the mutation result in different phenotypic consequences. CD45E613R B6 and 129 mice develop normally, while CD45E613R B6-129 F1 mice develop double stranded (ds)DNA autoantibodies, lymphoproliferative disease (LPD) and GN. CD45E613R BALB/c mice develop dsDNA autoantibodies, slight LPD, and no GN. This indicated genetic modifiers on these backgrounds modulated disease phenotype, and a single nucleotide polymorphism (SNP) screen performed between B6 and BALB/c mice for the development of ANAs identified two susceptibility loci. One locus, termed Wedge Associated Modifier (Wam) 1 on the BALB/c background identified tlr9 as the putative modifier gene. The second locus identified on the B6 background corresponded to the MHC H2 region. Here we examine the role of these two susceptibility regions in the CD45E613R BALB/c and B6 models of SLE. Previous studies from our lab identified two alleles of TLR9, the B6 (TLR9B6) and BALB/c (TLR9Ba) alleles, hypothesized to result in the signaling difference observed between the two backgrounds. In CD45E613R BALB/c TLR9-/- mice, B cell expression of CD45E613R and TLR9 is required for ANA development. Interestingly, CD45E613R B6 TLR9-/- mice acquired ANA development, suggesting the two alleles are regulating ANA and B cell tolerance in opposing ways. In Chapter II, we developed TLR9 congenic mice on the B6 and BALB/c backgrounds with the CD45E613R polymorphisms to determine if signaling difference and autoantibody development observed in intact mice were intrinsic to the TLR9 alleles. We show here that the TLR9Ba allele positively regulates autoreactive B cell development and is required for the development of ANAs, GN, and results in hyperresponsive B cell activation upon stimulation resistant CD45E613R B6 mice. Furthermore, the TLR9B6 allele negatively regulates ANA production, resulting in tolerance in susceptible CD45E613R BALB/c mice. The TLR9 alleles also dictate the signaling differences, such that the TLR9B6 allele results in hyperresponsive signaling and the TLR9Ba allele results in hyporesponsive signaling, regardless of genetic background, potentiating the differences in disease phenotypes. Together, these data demonstrate the dual role two allelic variants of TLR9 plays in regulating B cell tolerance. In Chapter III, we determine the role of the B6 susceptibility loci, MHC H2b (MHCB6), in the development of pathogenesis in the CD45E613R BALB/c model. We hypothesized the protective endogenous BALB/c MHC allele was preventing development of the severe GN observed in the CD45E613R B6-129 F1 model. In order to test this, we developed congenic CD45E613R BALB/c mice congenic for the MHC H2 region. CD45E613R BALB/c MHCB6 mice developed accelerated autoantibody production compared to CD45E613R BALB/c mice and eventually GN upon aging. B cells from CD45E613R BALB/c MHCB6 mice became dysregulated during development and were hyperactivated upon stimulation compared to CD45E613R BALB/c B cells. Further investigation revealed the B6 MHC H2b allele resulted in increased MHC class II expression on the surface of B cells compared to the BALB/c MHC H2d allele, but not other cell populations prior to disease onset. These data demonstrate one mechanism by which MHC susceptibility alleles identified in autoimmune patient populations are required for disease progression from autoreactivity to autoimmunity. Taken together with the TLR9 studies, we hypothesize the MHCB6 allele, in cooperation with the TLR9Ba allele and the sensitizing CD45E613R mutation results in the severe progression of autoimmunity in the CD45E613R B6 TLR9Ba model.

One of the hallmarks of the autoimmune disease systemic lupus erythematosus (SLE) is the production of pathogenic anti-nuclear antibodies (ANA). These autoantibodies are the result of a break in tolerance that allows for the activation and differentiation of autoreactive B cells into antibody-secreting plasma cells. ANA can form immune complexes with self nucleic acids, which can bind to and deposit in small blood vessels and instigate immunopathology. The CD45E613R knock-in model of SLE causes different phenotypic consequences on several murine genetic backgrounds, indicating that this signaling mutation is sensitive to genetic modifiers. Despite similar dysregulation of phosphatase activity, CD45E613R mice on a C57BL/6 (B6) background are resistant to SLE phenotypes, while CD45E613R.BALB/c are sensitive to ANA but not end organ damage. In an unbiased screen for genetic modifiers of ANA, we identify TLR9 as a putative modifier for the production of a specific ANA subtype, anti-dsDNA, in the context of CD45E613R.Here, we examine whether TLR9 modifies autoantibody production in the CD45E613R model between the resistant CD45E613R.B6 and sensitive CD45E613R.BALB/c model. Since CD45E613R alters Src family kinase and immunoreceptor signaling in several immune lineages, we generated a series of mixed bone marrow chimeras in the sensitive BALB/c genetic background to test which lineages require CD45E613R. TLR9 is also broadly expressed, so we examined which cell lineages require TLR9 and whether CD45E613R and TLR9 are necessary in cis or trans for anti-dsDNA IgG production in the BALB/c background. Upon finding that both CD45E613R and TLR9 are necessary in B cells for autoreactivity, we examined B cells from both genetic backgrounds. TLR9 stimulation induces elevated signaling in B cells from B6 mice compared to BALB/c, and these signals are not mediated by CD45E613R or co-stimulation via the BCR. We find that TLR9 negatively regulates ANA production in resistant CD45E613R.B6 mice by altering central B cell tolerance. In contrast, TLR9 positively regulates ANA production in BALB/c mice, likely because the strength of signal downstream of TLR9 is not sufficient for tolerance. These results indicate that TLR9 is a genetic modifier of autoreactivity in the context of dysregulated CD45 signaling.

Hypersensitivity pneumonitis (HP) is an interstitial lung disease caused by repeated inhalation of environmental antigens. The disease is characterized by alveolitis and granuloma formation; however, some patients develop chronic HP (CHP), a restrictive lung disease characterized by fibrosis. Previous studies revealed that neutrophils are recruited into the lung via a MyD88- dependent pathway and regulate disease severity through cytokine production. Toll-like receptors (TLRs) 2 and 9 recognize conserved molecular patterns present in bacteria, and signal through the adaptor molecule MyD88. The goal of my project is to investigate the role of TLRs 2 and 9 in the pathogenesis of HP during the acute phase, granulomatous phase, and chronic phase of the disease. Using the S. rectivirgula (SR) animal model of HP, our studies indicated that individually, TLRs 2 and 9 contributed to neutrophil recruitment. We generated TLR2/9 double knockout (TLR2/9−/− ) mice to determine the extent to which TLRs 2 and 9 cooperate in neutrophil recruitment during HP. During the acute response, TLR2/9−/− mice exposed to SR demonstrated a significant reduction in neutrophil recruitment after a single exposure to SR compared to C57BL/6 mice (WT). The reduction in neutrophils was associated with a reduction in the expression of the neutrophil chemokine CXCL2 and inflammatory cytokines TNFα and IL-6 in the BALF. Disease severity in HP is associated with granuloma formation and a Th17 response. After 3 weeks exposure to SR, our results demonstrate a decrease in IL-17 and IL-22 mRNA expression and a corresponding decrease in the percentage of Th17 cells in TLR2/9−/− mice compared to WT and single knockout (SKO) mice. The decrease in Th17 cells in TLR2/9−/− mice was not associated with a significant increase in T regulatory (Treg) cells or a switch to a Th1 response. In addition, TLR2−/− and TLR2/9−/− mice have significantly fewer activated CD4+ Th cells. Interstitial macrophages from TLR2/9−/− mice have decreased costimulatory molecule and MHCII expression suggesting they are deficient in T cell activation. However, TLR2−/− , TLR9−/− , and TLR2/9−/− still form granulomas in the lung. To determine the extent to which TLRs 2 and 9 contributed to the development of CHP, WT and TLR2/9−/− mice were intranasally exposed to SR three times/week for 15 weeks. WT and TLR2/9−/− mice developed a neutrophilic and lymphocytic alveolitis, but TLR2/9−/− mice had a significant increase in eosinophils in the bronchoalveolar lavage (BAL) fluid compared to WT exposed mice. Th17 cells, but not Th2 cells were detected in the lungs of mice exposed to SR for 14 weeks, suggesting CHP is not associated with a switch to a Th2 type immune response. WT and TLR2/9−/− mice had significantly reduced static compliance compared to WT unexposed mice. To determine whether the restrictive lung defect was associated with fibrosis, we visualized collagen in the lung by histology using Masson’s trichrome stain. Measuring collagen staining by positive pixel analysis suggested TLR2/9−/− mice were partially protected from lung fibrosis compared to WT mice. Altogether, the results suggest TLRs 2 and 9 cooperate in neutrophil recruitment and subsequently disease severity and play an integral role in the outcome of HP.

Asthma is a major cause of morbidity worldwide and this disease continues to increase in severity and prevalence, especially in the developed world. Clinical studies involving CpG-ODN therapy in asthmatics have identified that CpG-ODN drives a Th1 response, but does not inhibit airway hyperresponsiveness (AHR) in clinical asthma. In a murine fungal model of asthma, TLR9 plays a critical role in antifungal responses and is necessary for effective CpG-ODN therapy of asthma. Our experimental investigations have demonstrated that CpG-ODN administration promotes elevations in IL-12 levels, but no decrease in AHR in fungal asthma. These findings have provided a model in which to explore the mechanisms that inhibit CpG-ODN therapy from having efficacy for asthma. The IL-1R family protein ST2L is the receptor for IL-33. ST2L is present on immune and non-immune cells particularly during Th2-mediated disease. Recently, ST2L was identified as a negative regulator of MyD88-mediated TLR signaling. We have observed through immunohistochemical staining that ST2L progressively increases in the lung in asthmatic C57BL/6 mice along with the levels of TLR9. Herein, we have investigated whether blockade of ST2L enhances the therapeutic properties of CpG-ODN in a chronic fungal model of asthma. In our asthma model, C57BL/6 mice are sensitized to soluble A. fumigatus antigens and challenged with live A. fumigatus conidia. We investigated our model of fungal asthma first in TLR9+/+ and TLR9-/- mice followed by site directed therapy through either the intranasal (IN) or systemic intraperitoneal (IP) route from days 14 to 28-post conidia challenge. Additionally, mice were treated with an antibody targeting ST2L, IP CpG, IgG control, or combinations of the antibodies with CpG from days 14 to 28-post conidia challenge. Airway responses were attenuated in mice treated with α-ST2L+CpG, but not α-ST2L alone or CpG alone. In vitro studies with bone marrow derived DCs showed that targeting ST2L with CpG therapy decreased pSTAT3 levels in these cells, while increasing IL-12p70 levels. Together these data demonstrate that systemic TLR9 activation can reverse all features of chronic fungal asthma when delivered concomitantly with an anti-ST2L antibody. These data suggest that ST2L blockade enhances CpG-mediated therapeutic effects in asthma.

Abstract: Inflammatory Bowel Diseases (IBD) result from the nexus of a genetic predisposition, dysregulated immunologic insult against commensal microflora, and an environmental trigger. The intestinal epithelium is a single cell layer that separates a highly active mucosal immune system from a large antigenic load in the intestinal lumen. Innate immune recognition combined with a highly regulated adaptive immune response maintains this tolerance. The intestinal epithelium in collusion with antigen presenting cells primarily modulates this activity. In this thesis, we show that, in response to DNA isolated from bacteria, innate toll like receptor 9 (TLR9) activation in intestinal epithelial cells modulates both arms of the immune system, to regulate intestinal homeostasis, and through this mechanism, Bifidobacteria breve DNA exerts its anti-inflammatory function.

(PROBLEM) The overall goal of this thesis is to identify polymorphic alleles that associate with elevated risk and disease progression. Two different approaches were used to achieve this goal. (METHODS AIM 1) A database resource called Delta-MATCH was created using a predictive computational approach. The aim of the Delta-MATCH program is to identify human polymorphic variants that may create allele-specific transcription factor binding sites. In this version (v 1.0) 4,547,844 high-value candidate polymorphisms have been scored and ranked by the Delta-MATCH algorithm. These polymorphisms were either positioned within a 10,000 base pair window of a refSeq gene, or located within a region of high conservation in the human genome. The major and minor alleles for each of these 4.5 million polymorphisms were independently evaluated by the MATCH algorithm against a library of 550 known transcription factor binding site motifs (BIOBASE TRANSFAC v10.2) to determine the "highest MATCH scores" for each allele and transcription factor pair. (CONCLUSIONS AIM 1) The ranked list of Delta-MATCH predictions for each transcription factor binding site (matrix name) can be queried online at http://deltamatch.org. Predictions have been ranked in descending order of importance by a statistic called the "Delta-MATCH potential score", which reflects the potential of a polymorphism to create an allele-specific transcription factor binding site. (METHODS AIM 2) The common genotypes and haplotypes of four candidate genes (CCR5, TLR9, IRF5, APOE) were investigated for their association with the phenotype of HIV-1 viremia levels in a population of HIV-infected Americans primarily derived from the San Francisco SCOPE cohort. (CONCLUSIONS AIM 2) TLR9 and IRF5 variants associated with HIV viremia levels in White Americans. Additionally, individuals infected with HIV should try to avoid chronic inflammation, which means avoiding other viral and bacteria coinfections, traumas, and other behaviors that promote a chronic inflammatory state. Furthermore, the magnitude of TLR9- and IRF5-dependant inflammatory responses during the acute phase of HIV-1 infection may partially determine the viremia level of chronic infection (CVL classification).