The Role of Macrophages in the Response to TNF Inhibition in Experimental Arthritis.

The reduction of synovial tissue macrophages is a reliable biomarker for clinical improvement in patients with rheumatoid arthritis (RA), and macrophages are reduced in synovial tissue shortly after initiation of TNF inhibitors. The mechanism for this initial response is unclear. These studies were performed to identify the mechanisms responsible for the initial reduction of macrophages following TNF inhibition, positing that efflux to draining lymph nodes was involved. RA synovial tissue and synovial fluid macrophages expressed CCR7, which was increased in control macrophages following incubation with TNF-α. Human TNF transgenic (hTNF-Tg) mice were treated with infliximab after development of arthritis. Ankles were harvested and examined by histology, immunohistochemistry, quantitative RT-PCR, ELISA, and flow cytometry. hTNF-Tg mice treated with infliximab demonstrated significant clinical and histologic improvement 3 d after the initiation of therapy, at which time Ly6C+ macrophages were significantly reduced in the ankles. However, no evidence was identified to support a role of macrophage efflux to draining lymph nodes following treatment with infliximab. In contrast, apoptosis of Ly6C+ macrophages in the ankles and popliteal lymph nodes, decreased migration of monocytes into the ankles, and a reduction of CCL2 were identified following the initiation of infliximab. These observations demonstrate that Ly6C+ macrophage apoptosis and decreased ingress of circulating monocytes into the joint are responsible for the initial reduction of macrophages following infliximab treatment in hTNF-Tg mice.

Mechanisms regulating the loss of Tregs in HUPO mice that develop spontaneous inflammatory arthritis.

T regulatory cells (Tregs) are a potential therapeutic target in many autoimmune diseases including rheumatoid arthritis (RA). The mechanisms responsible for the maintenance of Tregs in chronic inflammatory conditions such as RA are poorly understood. We employed our mouse model of RA in which, the following deletion of Flice-like inhibitory protein in CD11c+ cells, CD11c-FLIP-KO (HUPO) mice develop spontaneous, progressive, erosive arthritis, with reduced Tregs, and the adoptive transfer of Tregs ameliorates the arthritis. HUPO thymic Treg development was normal, but peripheral of Treg Foxp3 was diminished mediated by reduction of dendritic cells and interleukin-2 (IL-2). During chronic inflammatory arthritis Tregs fail to maintain Foxp3, leading to non-apoptotic cell death and conversion to CD4+CD25+Foxp3- cells. Treatment with IL-2 increased Tregs and ameliorated the arthritis. In summary, reduced dendritic cells and IL-2 in the milieu of chronic inflammation, contribute to Treg instability, promoting HUPO arthritis progression, and suggesting a therapeutic approach in RA.

Critical role of synovial tissue-resident macrophage niche in joint homeostasis and suppression of chronic inflammation.

Little is known about the mechanisms regulating the transition of circulating monocytes into pro- or anti-inflammatory macrophages in chronic inflammation. Here, we took advantage of our novel mouse model of rheumatoid arthritis, in which Flip is deleted under the control of a CD11c promoter (HUPO mice). During synovial tissue homeostasis, both monocyte-derived F4/80int and self-renewing F4/80hi tissue-resident, macrophage populations were identified. However, in HUPO mice, decreased synovial tissue-resident macrophages preceded chronic arthritis, opened a niche permitting the influx of activated monocytes, with impaired ability to differentiate into F4/80hi tissue-resident macrophages. In contrast, Flip-replete monocytes entered the vacated niche and differentiated into tissue-resident macrophages, which suppressed arthritis. Genes important in macrophage tissue residency were reduced in HUPO F4/80hi macrophages and in leukocyte-rich rheumatoid arthritis synovial tissue monocytes. Our observations demonstrate that the macrophage tissue-resident niche is necessary for suppression of chronic inflammation and may contribute to the pathogenesis of rheumatoid arthritis.

Long-term stability of maxillary and mandibular arch dimensions when using rapid palatal expansion and edgewise mechanotherapy in growing patients.

OBJECTIVE: The purpose of this study was to assess the long-term stability of rapid palatal expansion (RPE) followed by full fixed edgewise appliances. METHODS: This study included 67 patients treated using Haas-type RPE and non-extraction edgewise appliance therapy at a single orthodontic practice. Serial dental casts were obtained at three different time points: pretreatment (T1), after expansion and fixed appliance therapy (T2), and at long-term recall (T3). The mean duration of the T1-T2 and T2-T3 periods was 4.8 ± 3.5 years and 11.0 ± 5.4 years, respectively. The dental casts were digitized, and the computed measurements were compared with untreated reference data. RESULTS: The majority of treatment-related increases in the maxillary and mandibular arch measurements were statistically significant (p < 0.05) and greater than expected for the untreated controls. Although many measurements decreased postretention (T2-T3), the net gains persisted for all of the measurements evaluated. CONCLUSIONS: The use of RPE therapy followed by full fixed edgewise appliances is an effective method for increasing maxillary and mandibular arch width dimensions in growing patients.

SNAPIN is critical for lysosomal acidification and autophagosome maturation in macrophages.

We previously observed that SNAPIN, which is an adaptor protein in the SNARE core complex, was highly expressed in rheumatoid arthritis synovial tissue macrophages, but its role in macrophages and autoimmunity is unknown. To identify SNAPIN's role in these cells, we employed siRNA to silence the expression of SNAPIN in primary human macrophages. Silencing SNAPIN resulted in swollen lysosomes with impaired CTSD (cathepsin D) activation, although total CTSD was not reduced. Neither endosome cargo delivery nor lysosomal fusion with endosomes or autophagosomes was inhibited following the forced silencing of SNAPIN. The acidification of lysosomes and accumulation of autolysosomes in SNAPIN-silenced cells was inhibited, resulting in incomplete lysosomal hydrolysis and impaired macroautophagy/autophagy flux. Mechanistic studies employing ratiometric color fluorescence on living cells demonstrated that the reduction of SNAPIN resulted in a modest reduction of H+ pump activity; however, the more critical mechanism was a lysosomal proton leak. Overall, our results demonstrate that SNAPIN is critical in the maintenance of healthy lysosomes and autophagy through its role in lysosome acidification and autophagosome maturation in macrophages largely through preventing proton leak. These observations suggest an important role for SNAPIN and autophagy in the homeostasis of macrophages, particularly long-lived tissue resident macrophages.

Association of Increased F4/80high Macrophages With Suppression of Serum-Transfer Arthritis in Mice With Reduced FLIP in Myeloid Cells.

OBJECTIVE: Macrophages are critical in the pathogenesis of rheumatoid arthritis (RA). We recently demonstrated that FLIP is necessary for the differentiation and/or survival of macrophages. We also showed that FLIP is highly expressed in RA synovial macrophages. This study was undertaken to determine if a reduction in FLIP in mouse macrophages reduces synovial tissue macrophages and ameliorates serum-transfer arthritis. METHODS: Mice with Flip deleted in myeloid cells (Flipf/f LysMc/+ mice) and littermate controls were used. Arthritis was induced by intraperitoneal injection of K/BxN serum. Disease severity was evaluated by clinical score and change in ankle thickness, and joints were examined by histology and immunohistochemistry. Cells were isolated from the ankles and bone marrow of the mice and examined by flow cytometry, real-time quantitative reverse transcriptase-polymerase chain reaction, or Western blotting. RESULTS: In contrast to expectations, Flipf/f LysMc/+ mice developed more severe arthritis early in the clinical course, but peak arthritis was attenuated and the resolution phase more complete than in control mice. Prior to the induction of serum-transfer arthritis, the number of tissue-resident macrophages was reduced. On day 9 after arthritis induction, the number of F4/80high macrophages in the joints of the Flipf/f LysMc/+ mice was not decreased, but increased. FLIP was reduced in the F4/80high macrophages in the ankles of the Flipf/f LysMc/+ mice, while F4/80high macrophages expressed an antiinflammatory phenotype in both the Flipf/f LysMc/+ and control mice. CONCLUSION: Our observations suggest that reducing FLIP in macrophages by increasing the number of antiinflammatory macrophages may be an effective therapeutic approach to suppress inflammation, depending on the disease stage.

CD11c-mediated deletion of Flip promotes autoreactivity and inflammatory arthritis.

Dendritic cells (DCs) are critical for immune homeostasis. To target DCs, we generated a mouse line with Flip deficiency in cells that express cre under the CD11c promoter (CD11c-Flip-KO). CD11c-Flip-KO mice spontaneously develop erosive, inflammatory arthritis, resembling rheumatoid arthritis, which is dramatically reduced when these mice are crossed with Rag(-/-) mice. The CD8α(+) DC subset is significantly reduced, along with alterations in NK cells and macrophages. Autoreactive CD4(+) T cells and autoantibodies specific for joint tissue are present, and arthritis severity correlates with the number of autoreactive CD4(+) T cells and plasmablasts in the joint-draining lymph nodes. Reduced T regulatory cells (Tregs) inversely correlate with arthritis severity, and the transfer of Tregs ameliorates arthritis. This KO line identifies a model that will permit in depth interrogation of the pathogenesis of rheumatoid arthritis, including the role of CD8α(+) DCs and other cells of the immune system.