1. Sean Stoessel MCB 5255

2. Ankylosing Spondylitis (AS) Chronic inflammation of the joints of the axial skeleton, including facet joints of spine and sacroiliac joint in hip. Fibrocartilage of joint is primary site of inflammation, but underlying bone often undergoes structural changes over time. Other features: IBD, peripheral arthritis, enthesitis, eye inflammation, carditis (rare) Effects 5 out of 1,000 adults 1 40% – 50% AS patients have HLA-B27 2 TNFα blockers commonly used treatment.

3. Common Sites of Inflammation Intervertebral joints (Facet joints) Sacroiliac Joint Gray (1918). Anatomy of the Human Body

4. Monocyte Activation and Differentiation Macrophages implicated in AS pathogenesis – histological evidence from human joint biopsies, gene expression analysis of cultured macrophages from patient PBMCs, work in animal models Osteoclasts also implicated, but involvement in Rheumatoid Arthritis better characterized. Macrophages and osteoclasts share cell lineage. Jeffrey W. Pollard. “Trophic macrophages in development and disease” Nature Reviews Immunology 9, 259-270 (April 2009)

5. TLR-mediated Activation of monocytes Monocyte recognition of PAMPs can cause up-regulation of pro-inflammatory cytokines Keep in mind for later: similarities between monocyte response to ER stress and TLR activation. Introduction to the Immune Sytem: Part II. What-when-how.com

6. In vivo pre-activation of monocytes in patients with axial spondyloarthritis Arthritis Research and Therapy 2015 Kristina Conrad, Peihua Wu, Joachim Sieper and Uta Syrbe

7. Figure 1. Conrad et al. “In vivo pre-activation of monocytes in patients with axial spondyloarthritis.” Arthritis Research and Therapy (2015) 17:179 Flow Cytometry data shows 3 distinct types of monocytes: Classical, intermediate, and non-classical. SpA patients show a higher proportion of classical monocytes in circulation.

8. Figure 1. Conrad et al. “In vivo pre-activation of monocytes in patients with axial spondyloarthritis.” Arthritis Research and Therapy (2015) 17:179

9. Figure 2. Conrad et al. “In vivo pre-activation of monocytes in patients with axial spondyloarthritis.” Arthritis Research and Therapy (2015) 17:179 A subset of SpA patients show elevated % of monocytes producing IL-β1, IL-6, and TNFα. % of IL-β1 producing monocytes increased most significantly after MDP or FSL treatment in SpA patients when compared to controls.

10. Figure 3. Conrad et al. “In vivo pre-activation of monocytes in patients with axial spondyloarthritis.” Arthritis Research and Therapy (2015) 17:179 A. IL-1 receptor antagonist expressing monocytes are elevated at baseline compared to control patients. No difference after incubation with PAMPs. B. IL-10 not upregulated in SpA patients as robustly in response to LPS. - Take away: Possible dysregulation of proinflammatory repsonse, but only in response to activation of a subset of TLRs?

11. Figure 4. Conrad et al. “In vivo pre-activation of monocytes in patients with axial spondyloarthritis.” Arthritis Research and Therapy (2015) 17:179 Take away: Separating SpA patients into conventional treatment and biologics treatment groups shows elevation in IL- β1, IL-6, and TNFα- producing monocytes only significant in conventional treatment group i.e. Not anti- TNF α antibody treated.

12. Summary and Remaining Questions Classical (CD14++, CD16-) monocytes more prevalent in SpA patients than control patients – Classical monocytes have been shown to have greater phagocytic activity and ROS production than non-classical population. A subset of the SpA patients show an increased % of IL-β1, IL-6, and TNFα-producing monocytes at baseline and a larger increase in the proportion of these monocytes than controls when exposed to FLS and MDP. This subset of patients are those treated conventionally with NSAIDs and not anti-TNFalpha biologics. In SpA patients, circulating monocytes may be more primed for a pro-inflammatory response, particularly when TNFalpha signaling has not been inhibited. Are bone marrow monocytes, destined to become osteoclasts, primed in the same way for differentiation and upregulation of pro-inflammatory cytokines? Is the priming cell-autonomous?

13. HLA-B27 Misfolding and the Unfolded Protein Response Rat model to be used has multiple transgene copies of HLA-B27 with multiple human beta2 microglobulin copies. Misfolding of HLA-B27 has been shown to cause UPR in macrophages and UPR signaling converges on TLR signaling pathways at TRAF activation (prior to NF-kB activation).

14. HLA–B27 Alters the Response to Tumor Necrosis Factor and Promotes Osteoclastogenesis in Bone Marrow Monocytes From HLA–B27–Transgenic Rats Gerlinde Layh-Schmitt, Eva Y. Yang, Grace Kwon, and Robert A. Colbert

15. Figure 1. Layh-Schmitt et al. “HLA–B27 Alters the Response to Tumor Necrosis Factor and Promotes Osteoclastogenesis in Bone Marrow Monocytes From HLA–B27–Transgenic Rats.” Arthritis and Rheumatism (2013) vol. 65, no. 8; pp 2123-2131 * RANKL and TNFalpha are known to stimulate osteoclast differentiation

16. Figure 2. Layh-Schmitt et al. “HLA–B27 Alters the Response to Tumor Necrosis Factor and Promotes Osteoclastogenesis in Bone Marrow Monocytes From HLA–B27–Transgenic Rats.” Arthritis and Rheumatism (2013) vol. 65, no. 8; pp 2123-2131 Take aways: 1)TNFα stimulates differentiation of more OCs in HLA- B27 monocytes but requires IL-1 α for this enhanced response. 2)Inhibition of IFN-β produces even greater osteoclastogenesis response in HLA- B27 monocytes.

17. Figure 3. Layh-Schmitt et al. “HLA–B27 Alters the Response to Tumor Necrosis Factor and Promotes Osteoclastogenesis in Bone Marrow Monocytes From HLA–B27–Transgenic Rats.” Arthritis and Rheumatism (2013) vol. 65, no. 8; pp 2123-2131 -Looking at effects of TNFα on HLA- B27 production and its misfolding. - TNFα, but not RANKL, will induce increase in misfolded HLA-B27, with subsequent upregulation of BiP and induction of the Unfolded Protein Response.

18. Figure 4. Layh-Schmitt et al. “HLA–B27 Alters the Response to Tumor Necrosis Factor and Promotes Osteoclastogenesis in Bone Marrow Monocytes From HLA–B27–Transgenic Rats.” Arthritis and Rheumatism (2013) vol. 65, no. 8; pp 2123-2131 Can ER stress alone increase IL-1 α and IFN-β expression? Yes, very strongly.

19. Figure 5. Layh-Schmitt et al. “HLA–B27 Alters the Response to Tumor Necrosis Factor and Promotes Osteoclastogenesis in Bone Marrow Monocytes From HLA–B27–Transgenic Rats.” Arthritis and Rheumatism (2013) vol. 65, no. 8; pp 2123-2131 Increase in staining for BiP and HLA- B27 seen after TNFα treatment and seems more apparent in monocytes rather than mature osteoclasts.

20. Summary and Remaining Questions TNFα, unlike RANKL, stimulates enhanced osteoclastogenesis in HLA-B27 rat monocytes and upregulates IL-1 α and IFN-β. These cytokines appear to be upregulated in response to the UPR, which TNF α triggers by increasing the levels of misfolded HLA-B27. The increased osteoclast differentiation in HLA-B27- expressing monocytes is likely a direct result of UPR signaling. Are other cytokines important in potentiating the inflammatory response and genes related to osteoblast signaling also dysregulated and are they only dysregulated in response to TNFα?

21. Bibliography Conrad et al. “In vivo pre-activation of monocytes in patients with axial spondyloarthritis.” Arthritis Research and Therapy (2015) 17:179 Layh-Schmitt et al. “HLA–B27 Alters the Response to Tumor Necrosis Factor and Promotes Osteoclastogenesis in Bone Marrow Monocytes From HLA–B27–Transgenic Rats.” Arthritis and Rheumatism (2013) vol. 65, no. 8; pp 2123-2131 Jeffrey W. Pollard. “Trophic macrophages in development and disease” Nature Reviews Immunology 9, 259-270 (April 2009) Gray, Henry. Anatomy of the Human Body. Philadelphia: Lea & Febiger, 1918; Wikipedia, “facet joint,” “sacroiliac joint.” Braem, Kristen and Rik J. Lories. “Insights into the pathophysiology of ankylosing spondylitis: Contributions from animal models” Joint Bone Spine 79 (2012) 243–248 Smith JA et al. Gene expression analysis of macrophages derived from ankylosing spondylitis patients reveals interferon-γ dysregulation” Arthritis and Rheumatism (2008) Volume 58, Issue 6; 1563–1888