Aberrant pro-inflammatory responses of CD20+ T cells in experimental arthritis.

CD20+ T cells comprise a highly inflammatory subset implicated in autoimmunity, including rheumatoid arthritis (RA). We sought to characterize the CD20+ T cell subset in the murine collagen-induced arthritis (CIA) model of RA and investigate the phenotype and functional relevance of CD3+CD20+ T cells in the lymph nodes and arthritic joints using flow cytometry and immunohistochemistry. We demonstrate that CD3+CD4+CD20+ and CD3+CD8+CD20+ T cells are expanded in the draining lymph nodes of CIA mice, produce increased levels of pro-inflammatory cytokines and are less susceptible to regulation by regulatory T cells. Notably, CD3+CD4+CD20+ and CD3+CD8+CD20+ T cells are enriched with CXCR5+PD-1+ T follicular helper cells and CXCR5-PD-1+ peripheral T helper cells, subsets of T cells implicated in promoting B-cell responses and antibody production within pathologically inflamed non-lymphoid tissues in RA. Our findings suggest CD20+ T cells are associated with inflammatory responses and may exacerbate pathology by promoting inflammatory B-cell responses.

JAK inhibitors disrupt T cell-induced proinflammatory macrophage activation.

OBJECTIVES: Macrophage subsets, activated by T cells, are increasingly recognised to play a central role in rheumatoid arthritis (RA) pathogenesis. Janus kinase (JAK) inhibitors have proven beneficial clinical effects in RA. In this study, we investigated the effect of JAK inhibitors on the generation of cytokine-activated T (Tck) cells and the production of cytokines and chemokines induced by Tck cell/macrophage interactions. METHODS: CD14+ monocytes and CD4+ T cells were purified from peripheral blood mononuclear cells from buffy coats of healthy donors. As representative JAK inhibitors, tofacitinib or ruxolitinib were added during Tck cell differentiation. Previously validated protocols were used to generate macrophages and Tck cells from monocytes and CD4+ T cells, respectively. Cytokine and chemokine including TNF, IL-6, IL-15, IL-RA, IL-10, MIP1α, MIP1ß and IP10 were measured by ELISA. RESULTS: JAK inhibitors prevented cytokine-induced maturation of Tck cells and decreased the production of proinflammatory cytokines TNF, IL-6, IL-15, IL-1RA and the chemokines IL-10, MIP1α, MIP1ß, IP10 by Tck cell-activated macrophages in vitro (p<0.05). CONCLUSIONS: Our findings show that JAK inhibition disrupts T cell-induced macrophage activation and reduces downstream proinflammatory cytokine and chemokine responses, suggesting that suppressing the T cell-macrophage interaction contributes to the therapeutic effect of JAK inhibitors.

Pro-inflammatory adiponectin in pediatric-onset multiple sclerosis.

BACKGROUND: Being obese is associated with both increased risk of developing multiple sclerosis (MS) and greater MS disease activity. OBJECTIVES: The objective of this study is to investigate levels and potential pathophysiologic contribution of serum adipose-hormones (adipokines) in pediatric-onset MS. METHODS: Following a Luminex adipokine screen, adiponectin (APN) and its isoforms were quantified by enzyme-linked immunosorbent assay (ELISA) in 169 children with incident acquired demyelinating syndromes (ADS), prospectively ascertained as having either MS or other forms of inflammatory central nervous system (CNS) demyelination. The effect of recombinant APN and APN-containing sera was assessed on functional responses of normal human peripheral blood myeloid and T cells and on human CNS-derived microglia. RESULTS: Compared to other cohorts, children with MS harbored higher serum APN levels, principally driven by higher levels of the low-molecular-weight isoform. Recombinant APN and pediatric MS serum-induced APN-dependent pro-inflammatory activation of CD14+ monocytes and of activated CD4+ and CD8+ T cells (both directly and indirectly through myeloid cells). APN induced human microglia activation while inhibiting their expression of molecules associated with quiescence. CONCLUSIONS: Elevated APN levels in children with MS may contribute to enhanced pro-inflammatory states of innate and adaptive peripheral immune responses and breach CNS-resident microglia quiescence, providing a plausible and potentially targetable mechanism by which APN contributes to MS disease activity.

Abnormal effector and regulatory T cell subsets in paediatric-onset multiple sclerosis.

Elucidation of distinct T-cell subsets involved in multiple sclerosis immune-pathophysiology continues to be of considerable interest since an ultimate goal is to more selectively target the aberrant immune response operating in individual patients. While abnormalities of both effector (Teff) and regulatory (Treg) T cells have been reported in patients with multiple sclerosis, prior studies have mostly assessed average abnormalities in either limb of the immune response, rather than both at the same time, which limits the ability to evaluate the balance between effectors and regulators operating in the same patient. Assessing both phenotypic and functional responses of Teffs and Tregs has also proven important. In studies of adults with multiple sclerosis, in whom biological disease onset likely started many years prior to the immune assessments, an added challenge for any reported abnormality is whether the abnormality indeed contributes to the disease (and hence of interest to target therapeutically) or merely develops consequent to inflammatory injury (in which case efforts to develop targeted therapies are unlikely to be beneficial). Paediatric-onset multiple sclerosis, though rare, offers a unique window into early disease mechanisms. Here, we carried out a comprehensive integrated study, simultaneously assessing phenotype and functional responses of both effector and regulatory T cells in the same children with multiple sclerosis, monophasic inflammatory CNS disorders, and healthy controls, recruited as part of the multicentre prospective Canadian Pediatric Demyelinating Disease Study (CPDDS). Stringent standard operating procedures were developed and uniformly applied to procure, process and subsequently analyse peripheral blood cells using rigorously applied multi-parametric flow cytometry panels and miniaturized functional assays validated for use with cryopreserved cells. We found abnormally increased frequencies and exaggerated pro-inflammatory responses of CD8+CD161highTCR-Vα7.2+ MAIT T cells and CD4+CCR2+CCR5+ Teffs in paediatric-onset multiple sclerosis, compared to both control groups. CD4+CD25hiCD127lowFOXP3+ Tregs of children with multiple sclerosis exhibited deficient suppressive capacity, including diminished capacity to suppress disease-implicated Teffs. In turn, the implicated Teffs of multiple sclerosis patients were relatively resistant to suppression by normal Tregs. An abnormal Teff/Treg ratio at the individual child level best distinguished multiple sclerosis children from controls. We implicate abnormalities in both frequencies and functional responses of distinct pro-inflammatory CD4 and CD8 T cell subsets, as well as Treg function, in paediatric-onset multiple sclerosis, and suggest that mechanisms contributing to early multiple sclerosis development differ across individuals, reflecting an excess abnormality in either Teff or Treg limbs of the T cell response, or a combination of lesser abnormalities in both limbs.

TLR2 stimulation regulates the balance between regulatory T cell and Th17 function: a novel mechanism of reduced regulatory T cell function in multiple sclerosis.

CD4(+)CD25(hi) FOXP3(+) regulatory T cells (Tregs) maintain tolerance to self-Ags. Their defective function is involved in the pathogenesis of multiple sclerosis (MS), an inflammatory demyelinating disease of the CNS. However, the mechanisms of such defective function are poorly understood. Recently, we reported that stimulation of TLR2, which is preferentially expressed by human Tregs, reduces their suppressive function and skews them into a Th17-like phenotype. In this study, we tested the hypothesis that TLR2 activation is involved in reduced Treg function in MS. We found that Tregs from MS patients expressed higher levels of TLR2 compared with healthy controls, and stimulation with the synthetic lipopeptide Pam3Cys, an agonist of TLR1/2, reduced Treg function and induced Th17 skewing in MS patient samples more than in healthy controls. These data provide a novel mechanism underlying diminished Treg function in MS. Infections that activate TLR2 in vivo (specifically through TLR1/2 heterodimers) could shift the Treg/Th17 balance toward a proinflammatory state in MS, thereby promoting disease activity and progression.

TLR2 stimulation drives human naive and effector regulatory T cells into a Th17-like phenotype with reduced suppressive function.

Naturally occurring CD4(+)CD25(+)FOXP3(+) regulatory T cells suppress the activity of pathogenic T cells and prevent development of autoimmune responses. There is growing evidence that TLRs are involved in modulating regulatory T cell (Treg) functions both directly and indirectly. Specifically, TLR2 stimulation has been shown to reduce the suppressive function of Tregs by mechanisms that are incompletely understood. The developmental pathways of Tregs and Th17 cells are considered divergent and mutually inhibitory, and IL-17 secretion has been reported to be associated with reduced Treg function. We hypothesized that TLR2 stimulation may reduce the suppressive function of Tregs by regulating the balance between Treg and Th17 phenotype and function. We examined the effect of different TLR2 ligands on the suppressive functions of Tregs and found that activation of TLR1/2 heterodimers reduces the suppressive activity of CD4(+)CD25(hi)FOXP3(low)CD45RA(+) (naive) and CD4(+)CD25(hi)FOXP3(hi)CD45RA(-) (memory or effector) Treg subpopulations on CD4(+)CD25(-)FOXP3(-)CD45RA(+) responder T cell proliferation while at the same time enhancing the secretion of IL-6 and IL-17, increasing RORC, and decreasing FOXP3 expression. Neutralization of IL-6 or IL-17 abrogated Pam3Cys-mediated reduction of Treg suppressive function. We also found that, in agreement with recent observations in mouse T cells, TLR2 stimulation can promote Th17 differentiation of human T helper precursors. We conclude that TLR2 stimulation, in combination with TCR activation and costimulation, promotes the differentiation of distinct subsets of human naive and memory/effector Tregs into a Th17-like phenotype and their expansion. Such TLR-induced mechanism of regulation of Treg function could enhance microbial clearance and increase the risk of autoimmune reactions.

Modulation of regulatory T cells in health and disease: role of toll-like receptors.

Naturally arising regulatory T cells (Tregs) originate from the thymus and are characterised by the expression of Foxp3 as a key control gene for their development and function. Their pivotal role is maintaining immunological self tolerance. Recently, Tregs have been shown to express Toll-like receptors (TLRs), which are essential components of the innate immune system for the detection of microbial infections and the activation of dendritic cells (DC) maturation programs to induce adaptive immune responses. TLRs are type 1 transmembrane receptors characterised by a highly variable extracellular region containing a leucine rich repeat domain (LRR) involved in ligand binding and an intracellular tail containing a highly conserved region, the TIR homology domain, which mediates interaction between TLRs and downstream signalling molecules. Recent data suggest that the activation of TLRs on Tregs can increase or decrease their suppressive activity, thus providing an important link between innate and adaptive immune responses. Treg modulation by TLRs might influence such processes as the response to infections, immune surveillance to cancer, transplant rejection, and the induction of autoimmunity. Understanding the link between Tregs and TLR could be beneficial to the discovery of new therapeutic targets and strategies.

Association between sputum smear status and local immune responses at the site of disease in HIV-infected patients with pulmonary tuberculosis.

Infection with human immunodeficiency virus (HIV) may affect the clinical presentation of pulmonary tuberculosis (TB). To investigate the association between sputum smear status at presentation and local pulmonary immune responses in HIV-infected patients with pulmonary TB, we compared the cellular and cytokine profiles in bronchoalveolar lavage (BAL) fluid obtained from the site of lung disease in 22 sputum smear- and culture-positive, and 17 sputum smear-negative but culture-positive pulmonary TB patients. Smear-positive patients had significantly higher BAL fluid concentrations of IL-6 (p=0.007), IL-8 (p=0.02), IL-10 (p=0.03) and IFN-gamma (p=0.008) than smear-negative patients. No significant differences in the proportions of examined BAL cells were found. We concluded that sputum smear-positive TB was associated with greater pro-inflammatory and immunomodulatory cytokine responses at the site of lung disease than sputum smear-negative disease. The local immune responses may affect the clinical presentation of active pulmonary TB in HIV-infected patients.

Alveolar macrophages from HIV-infected patients with pulmonary tuberculosis retain the capacity to respond to stimulation by lipopolysaccharide.

The functional capacity of alveolar macrophages (AM) in human immunodeficiency virus (HIV)-infected patients with pulmonary tuberculosis (TB) is not completely understood. To investigate the capacity of AM to mediate inflammatory responses, we obtained AM from human subjects by bronchoalveolar lavage (BAL) and studied the cells ex vivo. We compared AM from HIV-infected patients with suspected pulmonary TB to AM from healthy, HIV-negative controls for their capacity to produce TNF-alpha or IL-6 spontaneously and upon stimulation with lipopolysaccharide (LPS). Cytokine-producing cells were identified by macrophage markers and intracellular cytokine staining and flow cytometry. A higher proportion of AM from patients with microbiologically confirmed pulmonary TB than patients with probable TB or controls spontaneously expressed TNF-alpha shortly after isolation (geometric means: 38.5%, 23.7% and 15.8%, respectively), suggesting endogenous cytokine production. The proportions of AM spontaneously expressing TNF-alpha positively correlated with peripheral blood CD4(+) T-lymphocyte counts in patients (partial r=0.60, p=0.003) but not controls. Stimulation with LPS resulted in a significant increase in the proportions of TNF-alpha- and IL-6-positive AM from patients and controls (p<0.01). Bronchoalveolar lavage fluid (BALF) from confirmed TB patients also contained higher concentrations of the inflammatory cytokines predominantly produced by macrophages, IL-6 and IL-8, than controls (geometric mean cytokine concentrations per gram of BALF albumin were 1291 pg/g vs. 115 pg/g, p=0.03 for IL-6 and 4739 pg/g vs. 704 pg/g, p=0.03 for IL-8). We concluded that AM from HIV-infected patients with pulmonary TB produced and released inflammatory cytokines in vivo and retained their innate ability to respond to stimulation by LPS.

Mycobacterium tuberculosis resides in nonacidified vacuoles in endocytically competent alveolar macrophages from patients with tuberculosis and HIV infection.

Alveolar macrophages (AM) are the first professional phagocytes encountered by aerosols containing infections in the lungs, and their phagocytic capacity may be affected by these infections or environmental particles. The aim of this study was to evaluate the innate endocytic and phagocytic properties of human AM obtained from patients with pulmonary tuberculosis and to characterize the vacuoles in which Mycobacterium tuberculosis bacilli reside in vivo. AM were obtained by bronchoalveolar lavage from patients with suspected tuberculosis and from asymptomatic volunteers (controls). Clinical case definitions were based on mycobacterial culture of respiratory specimens and HIV serology. To assess phagocytosis, endocytosis, and acidification of the endosomal system, AM were cultured with IgG-coated polystyrene beads, dextran, and a pH-sensitive reporter (3-(2,4-dinitroanilino)-3-amino-N-methyldipropylamine) and were evaluated by light and immunoelectron microscopy. Cells from 89 patients and 10 controls were studied. We found no significant difference between the two groups in the ability of AM either to ingest beads and dextran or to deliver them to acidified lysosomes. In AM from patients with tuberculosis, the bacilli were located in vacuoles that failed to accumulate endocytosed material and were not acidified. We concluded that AM from patients with tuberculosis and HIV infections were competent to endocytose and phagocytose material and to deliver the material to functional, acidified lysosomes. M. tuberculosis residing in these AM arrests the progression of their phagosomes, which fail to fuse with acidified lysosomes. This confirms, for the first time in humans with tuberculosis and HIV, the conclusions from previous animal and in vitro studies.