Dissertation Defense: Regulating the Pathogenesis of Systemic Lupus Erythematosus through Lung Microbiome Manipulation, Myeloid-Specific STAT4 Knockout, and TLR9 Agonization

About this Dissertation

Lupus is an autoimmune disease with no known cure. There are a wide range of complex factors that affect severity, susceptibility, and disease presentation, which can include pulmonary and renal inflammation. As autoimmune disease prevalence rises globally, novel anti-inflammatory therapies for incurable, inflammatory diseases, such as systemic lupus erythematosus, are urgently needed. In order to find new therapeutics, however, a deeper and broader understanding of lupus pathogenesis and pathophysiology is required. This work is a compilation of one graduate student’s exploration of previously untouched areas, reexamination of discarded targets, and investigation into potential therapeutic pathways of lupus. While there is no cure that lies within, there is an attempt to both look broader and deeper at the complex web of factors that underlie this disease.

First, we describe the protective effect of intranasal antibiotic treatment (ABX) in the MRL/lpr lupus model. ABX-treated mice had decreased protein in their urine and decreased C3 and IgG deposition in the glomeruli, indicating less severe lupus nephritis. ABX-treated mice also had reduced lung infiltrating monocytes and T cells, as well as less IL-17 producing B and T cells in the lung-draining lymph nodes. Performing 16S rRNA sequencing, we found family-level differences in the bacterial composition of bronchoalveolar lavage fluid (BALF), but not in fecal samples. We found that bacterial families from the BALF correlated with local and regional disease parameters, while fecal bacterial families correlated with more distant inflammatory parameters, such as kidney size. We believe that we are the first group to do such an analysis into the role of the lung microbiome in lupus severity in humans or mice. We believe that the role of the lung microbiome in lupus and other inflammatory disorders deserves more attention as a possible route for therapeutic intervention.

Next, we will discuss the role of signal transducer and activator of transcription 4 (STAT4) signaling in lupus. Systemic lupus erythematosus is an autoimmune disease in which Type 1 and Type 3 immunity are pivotal. STAT4 is a Type 1 and Type 3 mediator, responding to both IL-12 and IL-23 to induce IFN-γ production. STAT4 SNPs have been associated with increased susceptibility to lupus in human subjects. Here, we describe how a LysM-Cre, Stat4-fl conditional knockout (KO) is protective in the MRL/lpr murine lupus model. KO mice had smaller spleens and lymph nodes, as well as reduced lupus nephritis. KO mice had a less inflammatory cellular makeup in their spleens and lymph nodes, which was partially recapitulated via in vitro studies with IL-12 and IL-23. Lastly, we found that the protection could not be replicated through the transfer of gut microbiome. We concluded that STAT4 is exacerbative in the MRL/lpr lupus model, suggesting it as a therapeutic target, however the exact mechanism behind that exacerbation is complex and requires further investigation.

Then we will delve into the first of two chapters that focus on the pattern recognition receptor, Toll-like receptor 9 (TLR9). TLR9 plays a crucial role in regulating lupus in both humans and lupus-prone MRL/lpr mice. Tlr9-/- MRL/lpr mice have exacerbated lupus symptoms. B cell-specific Tlr9-/- drives this increased disease severity, while knocking Tlr9 out of non-B cells has a minorly protective, but largely minimal, effect on lupus progression. Here we describe the potential protective effect of bacterial DNA (bDNA) in MRL/lpr mice. First, we demonstrate bDNA’s ability to induce IL-10 production and B regulatory cell (Breg) upregulation. We then describe two pathways by which this is achieved: one that relies on TLR9 signaling in B cells (B cell intrinsic) and another pathway that relies on TLR9 signaling in non-B cells (B cell extrinsic). We found that the B cell intrinsic pathway was IL-6 independent, while the B cell extrinsic pathway was IL-6 dependent. We then describe in vivo administration of bDNA in Tlr9+/+, Tlr9+/-, and Tlr9-/- mice. We found that bDNA was anti-inflammatory and protective in Tlr9+/- mice, however, in Tlr9+/+ mice, there was evidence of both anti-inflammatory and pro-inflammatory responses simultaneously. Ultimately, bDNA-treated Tlr9+/+ mice had worse lupus-like disease, despite an increase in Bregs. Our data suggests the importance, but also the complexity, of TLR9 signaling in lupus progression. Knowing that the B cell intrinsic pathway is protective, we foresee targeting this pathway for novel therapeutics against lupus.

Finally, we will discuss the therapeutic potential of a second TLR9 agonist, CpG-B oligodeoxynucleotides (ODN). Here, we show that a bDNA mimic, CpG-B ODN, activate TLR9 in a stronger manner, which leads to the exacerbation of lupus-like disease in mice, rather than protecting them. We find that treatment with these short DNA sequences increases the prevalence of inflammatory cells in the kidneys of treated mice, while also enhancing their proportion following in vitro stimulation. We then found that the effect of CpG-B ODN reverses and is mildly protective when Tlr9 is knocked out in myeloid cells, allowing for preferential activation of TLR9 in B cells. Overall, we find that TLR9 stimulation leads to pro- and anti-inflammatory effects, and that further research is needed to understand how that may the anti-inflammatory pathways could be harnessed into therapeutic benefit.

In summary, these four chapters range from hypothesis generating association data to deep investigations of individual receptors in lupus. These data may never yield a clinical benefit, but hopefully provide a deeper understanding of the different factors that contribute to lupus pathogenesis and pathophysiology.

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