A Case of Chronic Infectious Arthritis of the Temporomandibular Joint Associated with Osteomyelitis without Malocclusion

Infectious arthritis of the temporomandibular joint (TMJ) is rare, and previous reports have identified malocclusion resulting from condylar deformity and displacement of the condyle as one of the clinical characteristics of the disease. Here we report the case of a 33-year-old man with chronic infectious arthritis of the TMJ without malocclusion associated with osteomyelitis of the right mandible. Based on radiological findings of more prominent inflammation at the TMJ than in other regions and on the observed efficacy of antibiotic administration, we made a diagnosis of suppurative arthritis of the TMJ. Based on our empirical experience, including the present case, we speculate that refusal to cooperate with medical care may be a factor in the development of infectious arthritis of the TMJ

Expansion of Gal-9⁺ Th cells by exogenous Gal-9.

<p>(<b>A</b>) Naïve CD4 T cells were cultured under unstimulated, neutral, or Th17-skewing conditions for 4 days in the presence or absence of 30 nM human stable Gal-9 before surface Gal-9 expression was monitored by flow cytometry using an anti-mouse Gal-9 antibody. The antibody does not cross-react with the added human Gal-9. Dot plots are representative results obtained from neutral conditions in the presence or absence of exogenous Gal-9. (<b>B and C</b>) Flow cytometric analysis of Gal-9⁺ Th cell frequency after 4-day culture of naïve CD4 T cells under neutral conditions in the presence of blocking anti-IL-10 or anti-TGF-β antibody (<b>B</b>) or in the presence of the indicated concentration of IL-10 or 30 nM human stable Gal-9 (<b>C</b>). Results are means ± SEMs of quadruplicates. <i>p</i><0.001 (***), <i>p</i><0.01 (**), <i>p</i><0.05 (*), not significant (NS). Representative data out of 2 experiments are shown.</p

Identification of Gal-9⁺ Th cells.

<p>(<b>A</b>) Naïve CD4 T cells were cultured as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048574#pone-0048574-g001" target="_blank"><b>Figure 1A</b></a>, and cell-surface Gal-9 expression was monitored using flow cytometry. (<b>B</b>) Naïve CD4 T cells were sorted into Gal-9⁺ Th and Gal-9⁻ Th cells with a cell sorter and cultured under TCR stimulation or left unstimulated for 4 days before Gal-9 secretion into the culture media was measured. (<b>C</b>) Gal-9⁺ Th and Gal-9⁻ Th cells cultured under TCR stimulation for 4 days were examined for Gal-9 mRNA expression (left) and intracellular Gal-9 protein expression (right). (<b>D</b>) Cytokine mRNA expression in Gal-9⁺ Th and Gal-9⁻ Th cells cultured with or without TCR stimulation for 4 days. All the results are shown as the mean ± SEM of quadruplicates. <i>p</i><0.001 (***), <i>p</i><0.05 (*), not significant (NS). Representative data out of at least 2 experiments are shown.</p

Cell Surface Galectin-9 Expressing Th Cells Regulate Th17 and Foxp3⁺ Treg Development by Galectin-9 Secretion

<div><p>Galectin-9 (Gal-9), a β-galactoside binding mammalian lectin, regulates immune responses by reducing pro-inflammatory IL-17-producing Th cells (Th17) and increasing anti-inflammatory Foxp3⁺ regulatory T cells (Treg) <em>in vitro</em> and <em>in vivo</em>. These functions of Gal-9 are thought to be exerted by binding to receptor molecules on the cell surface. However, Gal-9 lacks a signal peptide for secretion and is predominantly located in the cytoplasm, which raises questions regarding how and which cells secrete Gal-9 <em>in vivo</em>. Since Gal-9 expression does not necessarily correlate with its secretion, Gal-9-secreting cells <em>in vivo</em> have been elusive. We report here that CD4 T cells expressing Gal-9 on the cell surface (Gal-9⁺ Th cells) secrete Gal-9 upon T cell receptor (TCR) stimulation, but other CD4 T cells do not, although they express an equivalent amount of intracellular Gal-9. Gal-9⁺ Th cells expressed interleukin (IL)-10 and transforming growth factor (TGF)-β but did not express Foxp3. In a co-culture experiment, Gal-9⁺ Th cells regulated Th17/Treg development in a manner similar to that by exogenous Gal-9, during which the regulation by Gal-9⁺ Th cells was shown to be sensitive to a Gal-9 antagonist but insensitive to IL-10 and TGF-β blockades. Further elucidation of Gal-9⁺ Th cells in humans indicates a conserved role of these cells through evolution and implies the possible utility of these cells for diagnosis or treatment of immunological diseases.</p> </div

Gal-9 secretion in naïve CD4 T cell culture upon TCR stimulation.

<p>(<b>A</b>) Naïve CD4 T cells were cultured in Th17-skewing conditions, neutral conditions (TCR stimulation), or without stimulation for 96 h before released Gal-9 was measured using ELISA. (<b>B</b>) Same as (<b>A</b>), except that the cells were cultured under Th17-skewing conditions, neutral conditions, neutral conditions plus TGF-β1 (TGF-β1 alone), or neutral conditions plus IL-6 (IL-6 alone). (<b>C</b>) Gal-9 mRNA was quantified using real-time RT-PCR. (<b>D</b>) Gal-9 secretion in the presence of human stable Gal-9 (30 nM) was measured. The human protein was not detected by mouse Gal-9 ELISA (see text for details). All results are shown as the mean ± SEM of quadruplicates. <i>p</i><0.001 (***), <i>p</i><0.05 (*), not significant (NS). Representative data out of at least 2 experiments are shown.</p

Phenotype of Gal-9⁺ Th cells.

<p>To clearly demonstrate the co-expression of cell-surface Gal-9 with either Foxp3 or Tim-3, highly sensitive Gal-9 staining with biotinylated-anti-Gal-9 antibody plus streptavidin-APC was employed. (<b>A</b>) Naïve CD4 T cells cultured under neutral conditions (left) or Treg-skewing conditions (right) for 4 days were examined for Foxp3 (Treg marker) and cell-surface Gal-9 expressions using flow cytometry. (<b>B</b>) Co-expression analysis of cell-surface Gal-9 and Tim-3 in naïve CD4 T cells cultured under neutral conditions for 4 days. (<b>C</b>) Intracellular IL-10 in naïve CD4 T cells purified from wild-type (WT) or Gal-9 knockout (Gal-9 KO) mice. (<b>D</b>) Naïve CD4 T cells from WT and Gal-9 KO mice were cultured under neutral or Th17-skewing conditions, and IL-10 mRNA expression was measured. Results are shown as the mean ± SEM of triplicate or quadruplicate. Symbol (*) represents significant (<i>p</i><0.05) differences from the indicated counterparts. Representative data out of at least 2 experiments are shown.</p

Regulatory function of Gal-9⁺ Th cells on Th17/Treg development.

<p>(<b>A–C</b>) Gal-9⁺ Th and Gal-9⁻ Th cells were obtained using a cell sorter from naïve CD4 T cells cultured under neutral conditions for 4 days. These cells were co-cultured with Th17-skewed cells at 1∶1 ratio and cultured for 90 h before analysis. (<b>A</b>) IL-17A and Foxp3 expression measured using ELISA and real-time RT-PCR, respectively. (<b>B</b>) IL-17A production in the Gal-9⁺ Th cell co-culture was examined in the presence of Gal-9 antagonist lactose or irrelevant sugar sucrose. (<b>C</b>) IL-17A production was examined in the presence of anti-IL-10 or anti-TGF-β blocking antibodies. (<b>D</b>) Naïve CD4 T cells were cultured under Th17-skewing conditions for 4 days in the presence of indicated concentration of IL-10 or human stable Gal-9 before IL-17A was measured. Results are shown as the mean ± SEM of quadruplicate experiments. <i>p</i><0.001 (***), <i>p</i><0.01 (**), <i>p</i><0.05 (*), not significant (NS). Representative data out of at least 2 experiments are shown.</p

Gal-9⁺ Th cells in humans.

<p>(<b>A</b>) CD4 T cells from human peripheral blood were cultured with or without TCR stimulation for 4 days. Surface Gal-9 and CD25 expression was analyzed using flow cytometry. Results are shown as the mean ± SEM from 4 healthy donors. (<b>B</b>) CD4 T cells were cultured under neutral conditions for 4 days and then sorted into Gal-9⁺ CD25⁺ Th cells and Gal-9⁻ CD25⁺ Th cells. Sorted cells were cultured under neutral conditions for 4 days before the measurement of secreted Gal-9 by ELISA or cytokine mRNA using real-time RT-PCR. Results are shown as the mean ± SEM from 8 healthy donors. * <i>p</i><0.05. Data from 2 representative experiments are shown.</p