NADPH Oxidase Modifies Patterns of MHC Class II–Restricted Epitopic Repertoires through Redox Control of Antigen Processing

Abstract

The chemistries within phagosomes of APCs mediate microbial destruction as well as generate peptides for presentation on MHC class II. The antimicrobial effector NADPH oxidase (NOX2), which generates superoxide within maturing phagosomes, has also been shown to regulate activities of cysteine cathepsins through modulation of the lumenal redox potential. Using real-time analyses of lumenal microenvironmental parameters, in conjunction with hydrolysis pattern assessment of phagocytosed proteins, we demonstrated that NOX2 activity not only affects levels of phagosomal proteolysis as previously shown, but also the pattern of proteolytic digestion. Additionally, it was found that NOX2 deficiency adversely affected the ability of bone marrow–derived macrophages, but not dendritic cells, to process and present the I-Ab–immunodominant peptide of the autoantigen myelin oligodendrocyte glycoprotein (MOG). Computational and experimental analyses indicated that the I-Ab binding region of the immunodominant peptide of MOG is susceptible to cleavage by the NOX2-controlled cysteine cathepsins L and S in a redox-dependent manner. Consistent with these findings, I-Ab mice that were deficient in the p47phox or gp91phox subunits of NOX2 were partially protected from MOG-induced experimental autoimmune encephalomyelitis and displayed compromised reactivation of MOG-specific CD4+ T cells in the CNS, despite eliciting a normal primary CD4+ T cell response to the inoculated MOG Ag. Taken together, this study demonstrates that the redox microenvironment within the phagosomes of APCs is a determinant in MHC class II repertoire production in a cell-specific and Ag-specific manner, which can ultimately impact susceptibility to CD4+ T cell–driven autoimmune disease processes.

Redundancy between Cysteine Cathepsins in Murine Experimental Autoimmune Encephalomyelitis

“…All animal work in this study was specifically approved (M11029) by the University of Calgary Animal Care and Use Committee. BMMØs were derived from bone marrow of 8–12 week old male mice using L-929-conditioned media as a source of M-CSF over a period of 10–14 days as previously described [30–34]. QPCR confirmed relative levels or absence of specific cathepsin mRNA in BMMØ cultures (S1 Protocol, S1 Fig).…”

“…To determine if BMMØs from cathepsin-deficient mice are capable of presenting murine MOG antigens to 2D2 CD4+ T cells, BMMØs derived from WT and cathepsin B, S, L, B/S, or S/L deficient mice were exposed to the immunodominant I-Ab epitope murine MOG 35-55 (0, 1, 10, 25 μg/ml; 0.39–9.7 nM), or to the full extracellular domain of murine MOG (MOG 1-125 ; 0, 1, 10, 25 μg/ml; 0.039–0.96 nM) [31, 34, 37, 38], for 6 h within culture medium. Cultures without BMMØs or MOG, or with concanavalin A (ConA, 5 μg/ml) treatment, were included as negative and positive controls for specific presentation and T cell proliferation respectively.…”

“…MOG 35-55 was synthesized by the University of Calgary Peptide Services, Calgary, AB, Canada. MOG 1-125 was prepared and characterized as previously described [34]. In brief, MOG 1-125 was cloned with a carboxy-terminal 6xHIStag, and expressed using Rosetta blue (DE3) E .…”

“…In brief, MOG 1-125 was cloned with a carboxy-terminal 6xHIStag, and expressed using Rosetta blue (DE3) E . coli [34]. The tagged protein was purified using Ni-NTA agarose beads (Qiagen).…”

Autophagy proteins are not universally required for phagosome maturation

“…Indeed, CYBB has a controversial role in modulating phagosome maturation; some investigators find that it delays phagosomal maturation, 23,24 whereas others do not. [25][26][27] Recent studies showed that the maturation status of macrophages dictates the effects that the CYBB NADPH oxidase has on phagosome maturation. 28 Based on these findings, we hypothesize that both CYBB and ATG proteins, which are linked in phagosomal development, may have effects on phagosome maturation that are condition-dependent and possibly dynamic-altered by status of the macrophages, e.g., in classically activated macrophages (M1) vs. alternatively activated macrophages (M2).…”