Traumatic brain injury (TBI) elicits innate inflammatory responses that can lead to secondary brain injury. To better understand the mechanisms involved in TBI‐induced inflammation, we examined the ...nature of macrophages responding to TBI in mice. In this model, brain macrophages were increased >20‐fold the day after injury and >77‐fold 4 days after injury in the ipsilateral hemisphere compared with sham controls. TBI macrophage subsets were identified by using a reporter mouse strain (YARG) that expresses eYFP from an internal ribosome entry site (IRES) inserted at the 3′ end of the gene for arginase‐1 (Arg1), a hallmark of alternatively activated (M2) macrophages. One day after TBI, 21 ± 1.5% of ipsilateral brain macrophages expressed relatively high levels of Arg1 as detected by yellow fluorescent protein, and this subpopulation declined thereafter. Arg1+ cells localized with macrophages near the TBI lesion. Gene expression analysis of sorted Arg1+ and Arg1− brain macrophages revealed that both populations had profiles that included features of conventional M2 macrophages and classically activated (M1) macrophages. The Arg1+ cells differed from Arg1− cells in multiple aspects, most notably in their chemokine repertoires. Thus, the macrophage response to TBI initially involves heterogeneous polarization toward at least two major subsets.
Dendritic cells (DCs), which are known to support immune activation during infection, may also regulate immune homeostasis in resting animals. Here we show that mice lacking the ubiquitin-editing ...molecule A20 specifically in DCs spontaneously showed DC activation and population expansion of activated T cells. Analysis of DC-specific epistasis in compound mice lacking both A20 and the signaling adaptor MyD88 specifically in DCs showed that A20 restricted both MyD88-independent signals, which drive activation of DCs and T cells, and MyD88-dependent signals, which drive population expansion of T cells. In addition, mice lacking A20 specifically in DCs spontaneously developed lymphocyte-dependent colitis, seronegative ankylosing arthritis and enthesitis, conditions stereotypical of human inflammatory bowel disease (IBD). Our findings indicate that DCs need A20 to preserve immune quiescence and suggest that A20-dependent DC functions may underlie IBD and IBD-associated arthritides.
While general population studies have shown inverse associations between physical activity and common inflammatory biomarkers, the effects of physical activity on inflammatory gene expression and ...signaling pathways in rheumatoid arthritis (RA) remain unknown. We aimed to determine whether physical activity independently associates with expression of inflammatory genes among people with RA.
This was a prospective observational study of adults with RA. Physical activity was measured by quantitative actigraphy over 7 consecutive days, and peripheral blood collected during the same time period was used for RNA sequencing followed by differential gene expression, pathway, and network analyses.
Actigraphy and RNA sequencing data were evaluated in 35 patients. The cohort had a mean age of 56 (SD 12) years, and was 91% female, 31% White, 9% Black, 9% Asian, and 40% Hispanic. We found 767 genes differentially expressed (adjusted
< 0.1) between patients in the greatest vs lowest physical activity tertiles, after adjusting for sex, age, race, and ethnicity. The most active patients exhibited dose-dependent downregulation of several immune signaling pathways implicated in RA pathogenesis. These included CD40, STAT3, TREM-1, interleukin (IL)-17A, IL-8, Toll-like receptor, and interferon (IFN) signaling pathways. Upstream cytokine activation state analysis predicted reduced activation of tumor necrosis factor-α and IFN in the most active group. In sensitivity analyses, we adjusted for RA disease activity and physical function and found consistent results.
Patients with RA who were more physically active had lower expression of immune signaling pathways implicated in RA pathogenesis, even after adjusting for disease activity, suggesting that physical activity may confer a protective effect in RA.
The immunoreceptor tyrosine‐based activation motif (ITAM) is a highly conserved region in the cytoplasmic domain of signaling chains and receptors and is a critical mediator of intracellular signals. ...ITAM‐mediated signals depend on the Syk or ζ‐associated protein of 70 kDa tyrosine kinases, and ITAM signaling is required for the differentiation and function of B and T cells in adaptive immunity. ITAM‐dependent receptors also regulate the function of innate immune cells, including natural killer cells, and myeloid‐derived cells such as macrophages, neutrophils, dendritic cells, and mast cells. Myeloid lineage cells also include osteoclasts (OCLs), the cells required for bone resorption, and recent studies show a critical role for the ITAM‐containing adapter proteins DAP12 and the FcRγ chain (Fcɛ receptor I γ chain) in OCL differentiation. Mice deficient in both the DAP12 and FcRγ ITAM‐bearing adapters are significantly osteopetrotic with a severe defect in OCL differentiation, demonstrating the requirement for ITAM signals in bone and further implicating this pathway in the development of highly specialized cell functions in hematopoietic cells. Regulation of osteoclastogenesis by ITAM‐dependent receptors suggests that OCLs, similar to related myeloid cells, are tightly controlled by arrays of receptors that allow them to sense and respond to their local microenvironment like other innate immune cells.
Nerve growth factor (NGF) is a neurotrophin that mediates pain sensitization in pathologic states, including osteoarthritis. In clinical trials, antibodies to NGF reduce pain and improve physical ...function due to osteoarthritis of the knee or hip and have a long duration of action. Rapidly progressive osteoarthritis is a dose-dependent adverse event with these agents, and additional joint safety signals, such as subchondral insufficiency fractures and increased rates of total joint replacement, are reported. The effects on pain and potential mechanisms behind these joint events both are of considerable importance in the consideration of future use of anti-NGF therapies for osteoarthritis.
Deficiency of the signaling adapter protein DAP12 or its associated receptor TREM2 is associated with abnormal OC development in humans. Here we examine the role of TREM2 in mouse OC development and ...function, including migration and resorption in vitro. These results provide new evidence that TREM2 regulates OC function independent of its effects on multinucleated OC differentiation.
Introduction: TREM2 (triggering receptor expressed in myeloid cells‐2) associates with the signaling adapter DAP12 in osteoclasts (OCs). Genetic mutation or deletion of either the TYROBP (DAP12) or TREM2 gene is associated with the human disorder of brain and bone, Nasu‐Hakola disease. We and others recently showed the critical requirement for immunoreceptor tyrosine‐based activation motif (ITAM) signals through DAP12 and the Fc Receptor γ chain (FcRγ) during OC development. Here, we further define the role of TREM2 in OC differentiation and describe a role for TREM2 in OC migration and bone resorption.
Materials and Methods: We generated monoclonal anti‐mouse TREM2 antibodies (mAb), analyzed pre‐osteoclasts and mature OCs for TREM2 surface expression, and determined the effect of antibody ligation on in vitro OC differentiation, resorption, and migration. TREM2 RNA interference (RNAi) was used to disrupt expression of TREM2 in pre‐osteoclasts.
Results: Using flow cytometry, our studies reveal that TREM2 is weakly expressed on C57BL/6 bone marrow macrophages (BMMs) and is upregulated during culture with RANKL and macrophage‐colony stimulating factor (M‐CSF). The expression of TREM2 is unaltered in DAP12‐deficient OCs. Using C57BL/6 BMMs or RAW264.7 precursors, anti‐TREM2 mAb treatment with RANKL and M‐CSF enhances the formation of multinuclear TRACP+ OCs compared with control mAb treatment. In contrast, these agents have no effect on DAP12‐deficient precursors. Monoclonal Ab blockade of TREM2 on OCs generated from C57BL/6 BMMs results in decreased resorption of artificial calcium‐phosphate substrate and dentine. Reduction of TREM2 expression in RAW264.7 cells by RNAi results in loss of OC formation in response to RANKL and M‐CSF. Anti‐TREM2 cross‐linking enhances migration of C57BL/6 OCs and RAW246.7 OCs in response to M‐CSF.
Conclusions: Our studies indicate that the TREM2 receptor regulates OC multinucleation as well as resorption and migration of mature OCs. Thus, TREM2‐DAP12 signals regulate both OC formation and function.
Immunoreceptor tyrosine-based activation motif (ITAM) signaling mediated by DAP12 or Fcepsilon receptor Igamma chain (FcRgamma) have been shown to be critical for osteoclast differentiation and ...maturation under normal physiological conditions. Their function in pathological conditions is unknown. We studied the role of ITAM signaling during rapid bone remodeling induced by acute estrogen-deficiency in wild-type (WT), DAP12-deficient (DAP12-/-), FcRgamma-deficient (FcRgamma-/-) and double-deficient (DAP12-/-FcRgamma-/-) mice. Six weeks after ovariectomy (OVX), DAP12-/-FcRgamma-/- mice showed resistance to lumbar vertebral body (LVB) trabecular bone loss, while WT, DAP12-/- and FcRgamma-/- mice had significant LVB bone loss. In contrast, all ITAM adapter-deficient mice responded to OVX with bone loss in both femur and tibia of approximately 40%, relative to basal bone volumes. Only WT mice developed significant cortical bone loss after OVX. In vitro studies showed microenvironmental changes induced by OVX are indispensable for enhanced osteoclast formation and function. Cytokine changes, including TGFbeta and TNFalpha, were able to induce osteoclastogenesis independent of RANKL in BMMs from WT but not DAP12-/- and DAP12-/-FcRgamma-/- mice. FSH stimulated RANKL-induced osteoclast differentiation from BMMs in WT, but not DAP12-/- and DAP12-/-FcRgamma-/- mice. Our study demonstrates that although ITAM adapter signaling is critical for normal bone remodeling, estrogen-deficiency induces an ITAM adapter-independent bypass mechanism allowing for enhanced osteoclastogenesis and activation in specific bony microenvironments.
Lung myeloid cells are important in pulmonary immune homeostasis and in the pathogenesis of chronic obstructive pulmonary disease (COPD). Multiparameter immunophenotypic characterization of these ...cells is challenging because of their autofluorescence and diversity. We evaluated the immunophenotypic landscape of airway myeloid cells in COPD using time of flight mass cytometry. Cells from BAL, which were obtained from never-smokers (
= 8) and smokers with (
= 20) and without (
= 4) spirometric COPD, were examined using a 44-parameter time of flight mass cytometry panel. Unsupervised cluster analysis was used to identify cellular subtypes that were confirmed by manual gating. We identified major populations of CD68
and CD68
cells with 22 distinct phenotypic clusters, of which 18 were myeloid cells. We found a higher abundance of putative recruited myeloid cells (CD68
classical monocytes) in BAL from patients with COPD. CD68
classical monocyte population had distinct responses to smoking and COPD that were potentially related to their recruitment from the interstitium and vasculature. We demonstrate that BAL cells from smokers and subjects with COPD have lower AXL expression. Also, among subjects with COPD, we report significant differences in the abundance of PDL1
and PDL2
clusters and in the expression of PDL1 and PDL2 across several macrophage subtypes suggesting modulation of inflammatory responses. In addition, several phenotypic differences in BAL cells from subjects with history of COPD exacerbation were identified that could inform potential disease mechanisms. Overall, we report several changes to the immunophenotypic landscape that occur with smoking, COPD, and past exacerbations that are consistent with decreased regulation and increased activation of inflammatory pathways.
T cell immunoglobulin-domain and mucin-domain (TIM) proteins constitute a receptor family that was identified first on kidney and liver cells; recently it was also shown to be expressed on T cells. ...TIM-1 and -3 receptors denote different subsets of T cells and have distinct regulatory effects on T cell function. Ferritin is a spherical protein complex that is formed by 24 subunits of H- and L-ferritin. Ferritin stores iron atoms intracellularly, but it also circulates. H-ferritin, but not L-ferritin, shows saturable binding to subsets of human T and B cells, and its expression is increased in response to inflammation. We demonstrate that mouse TIM-2 is expressed on all splenic B cells, with increased levels on germinal center B cells. TIM-2 also is expressed in the liver, especially in bile duct epithelial cells, and in renal tubule cells. We further demonstrate that TIM-2 is a receptor for H-ferritin, but not for L-ferritin, and expression of TIM-2 permits the cellular uptake of H-ferritin into endosomes. This is the first identification of a receptor for ferritin and reveals a new role for TIM-2.
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