TLR

1. TLR MYD88-DEPENDENT MYD88-INDEPENDENT TIR TIR TIR TIR Myd88 TRIF TIRAP TRAM TRAF6 IRF3 (+ phosphorylation) IκBα P NF-κB IκBα IRF3 NF-κB NUCLEAR TRANSLOCATION IMMUNE RESPONSE GENES TYPE I IFN IRF3 NF-κB NF-κB

2. Lee EG et al2000 Science: Failure to regulate TNF-induced NF-kappaB and cell death responses in A20-deficient mice. Boone DL et al2004 Nat Immunol: The ubiquitin-modifying enzyme A20 is required for termination of Toll-like receptor responses. Wertz IE et al2004 Nature: De-ubiquitination and ubiquitin ligase domains of A20 downregulate NF-kappaB signalling.

3. host cells (e.g. APCs) ”sense” microbial contents in homeostasis • by TLRs and non-TLRs (e.g. NOD-like receptors) via binding to PAMPs Why do homeostatic TLR signals not trigger inflammation? How immune responses to commensal microflora are restrained during homeostasis? • quantity or strength of TLR signals • cell-type specific responses • cellular location of PAMP/TLR interactions • additional extra- or intracellular cues (regulator proteins e.g. A20)

4. What is the role of the adaptor protein Myd88 in causing inflammation in A20-/- mice? → (i) Myd88 signals drive potent proinflammatory responses in the absence of A20 → (ii) A20 prevents recruitment of innate immune cells by restricting Myd88-dependent signals

5. Do basal Myd88-dependent signals cause spontaneous activation and expansion of T cells in A20-/- mice? → Homeostatic Myd88-dependent signals cause T cell expansion and activation in the absence of A20

6. Could the role of A20 involve signals on either HSCs or stromal cell? Or both? → Homeostatic Myd88 signals drive a broad spectrum of spontaneous proinflammatory pathways in the absence of A20 in HCs

7. Does the commensal flora stimulate the homeostatic Myd88-dependent signals causing inflammation in A20-/- mice? Abx: vancomycin neomycin metronidazole ampicillin trimethoprim-sulfamethoxazole Ctrl: trimethoprim-sulfamethoxazole → Commensal bacteria drive spontaneous inflammation via Myd88 signals in the absence of A20

8. How A20 regulates TRIF-dependent TLR responses? → (i) A20 is required for restricting acute – Myd88-independent – TRIF-dependent proinflammatory signals → (ii) Myd88- and IRF3-independent IFN-β production in the absence of A20

9. TRAF6 as a physiological target for A20 during TLR signaling → A20 is essential for restricting TRAF6 ubiquitylation

10. SUMMARY &DISCUSSION • Homeostatic TLR signals can in fact be inflammatory and rapidly lethal! ( ≤ BALANCE ≤ ) • A20 enzymatically regulates ubiquitylation of signaling proteins • (activity, stability, inhibiting further reactivation) • amplified effect by regulating other non-TLR signals, e.g. TNF • (but Myd88 dominance!) • intestinal bacteria are a major source of Myd88-dependent signals • role of A20 in epithelial cell function (mucosal DCs!)? • A20 restricts TRIF-dependent IFN responses by limiting TRIF-dependent NF-κB signaling • IL-10-/- with progressive colitis in 4-6 mo •  regulation of intestinal inflammation (STAT3) • A20-/- with widespread systemic inflammation in 3-6 wk •  regulation of systemic immune activation (TRAF6) IL-10 vs. A20

11. TLR MYD88-DEPENDENT TIR TIR TIR TIR Myd88 TRIF MYD88-INDEPENDENT TIRAP TRAM A20 TRAF6 A20 IκBα P NF-κB IκBα NF-κB NUCLEAR TRANSLOCATION IMMUNE RESPONSE GENES TYPE I IFN NF-κB NF-κB