Granulocyte-Macrophage Colony Stimulating Factor As an Indirect Mediator of Nociceptor Activation and Pain.

The interaction between the immune system and the nervous system has been at the center of multiple research studies in recent years. Whereas the role played by cytokines as neuronal mediators is no longer contested, the mechanisms by which cytokines modulate pain processing remain to be elucidated. In this study, we have analyzed the involvement of granulocyte-macrophage colony stimulating factor (GM-CSF) in nociceptor activation in male and female mice. Previous studies have suggested GM-CSF might directly activate neurons. However, here we established the absence of a functional GM-CSF receptor in murine nociceptors, and suggest an indirect mechanism of action, via immune cells. We report that GM-CSF applied directly to magnetically purified nociceptors does not induce any transcriptional changes in nociceptive genes. In contrast, conditioned medium from GM-CSF-treated murine macrophages was able to drive nociceptor transcription. We also found that conditioned medium from nociceptors treated with the well established pain mediator, nerve growth factor, could also modify macrophage gene transcription, providing further evidence for a bidirectional crosstalk.SIGNIFICANCE STATEMENT The interaction of the immune system and the nervous system is known to play an important role in the development and maintenance of chronic pain disorders. Elucidating the mechanisms of these interactions is an important step toward understanding, and therefore treating, chronic pain disorders. This study provides evidence for a two-way crosstalk between macrophages and nociceptors in the peripheral nervous system, which may contribute to the sensitization of nociceptors by cytokines in pain development.

TNF and granulocyte macrophage-colony stimulating factor interdependence mediates inflammation via CCL17.

TNF and granulocyte macrophage-colony stimulating factor (GM-CSF) have proinflammatory activity and both contribute, for example, to rheumatoid arthritis pathogenesis. We previously identified a new GM-CSF→JMJD3 demethylase→interferon regulatory factor 4 (IRF4)→CCL17 pathway that is active in monocytes/macrophages in vitro and important for inflammatory pain, as well as for arthritic pain and disease. Here we provide evidence for a nexus between TNF and this pathway, and for TNF and GM-CSF interdependency. We report that the initiation of zymosan-induced inflammatory pain and zymosan-induced arthritic pain and disease are TNF dependent. Once arthritic pain and disease are established, blockade of GM-CSF or CCL17, but not of TNF, is still able to ameliorate them. TNF is required for GM-CSF-driven inflammatory pain and for initiation of GM-CSF-driven arthritic pain and disease, but not once they are established. TNF-driven inflammatory pain and TNF-driven arthritic pain and disease are dependent on GM-CSF and mechanistically require the same downstream pathway involving GM-CSF→CCL17 formation via JMJD3-regulated IRF4 production, indicating that GM-CSF and CCL17 can mediate some of the proinflammatory and algesic actions of TNF. Given we found that TNF appears important only early in arthritic pain and disease progression, targeting a downstream mediator, such as CCL17, which appears to act throughout the course of disease, could be effective at ameliorating chronic inflammatory conditions where TNF is implicated.

Immune Cytokines and Their Receptors in Inflammatory Pain.

There is burgeoning interest in the interaction between the immune and nervous systems. Pain is mediated by primary sensory neurons (nociceptors) that can respond to a variety of thermal, mechanical and chemical signals. Cytokines are now recognized as important mediators of inflammatory pain. They can induce nociceptor sensitization indirectly via mediators, wherein neurons become primed and thus become more responsive to stimulation; alternatively, there is also evidence that cytokines can directly activate neurons via their specific receptors present on the neuronal cells. We review here the evidence for and against these respective mechanisms, focusing on arthritis and inflammatory skin models. A number of striking inconsistencies amongst the conclusions made in the literature are highlighted and discussed.

G-CSF Receptor Blockade Ameliorates Arthritic Pain and Disease.

G-CSF or CSF-3, originally defined as a regulator of granulocyte lineage development via its cell surface receptor (G-CSFR), can play a role in inflammation, and hence in many pathologies, due to its effects on mature lineage populations. Given this, and because pain is an extremely important arthritis symptom, the efficacy of an anti-G-CSFR mAb for arthritic pain and disease was compared with that of a neutrophil-depleting mAb, anti-Ly6G, in both adaptive and innate immune-mediated murine models. Pain and disease were ameliorated in Ag-induced arthritis, zymosan-induced arthritis, and methylated BSA/IL-1 arthritis by both prophylactic and therapeutic anti-G-CSFR mAb treatment, whereas only prophylactic anti-Ly6G mAb treatment was effective. Efficacy for pain and disease correlated with reduced joint neutrophil numbers and, importantly, benefits were noted without necessarily the concomitant reduction in circulating neutrophils. Anti-G-CSFR mAb also suppressed zymosan-induced inflammatory pain. A new G-CSF-driven (methylated BSA/G-CSF) arthritis model was established enabling us to demonstrate that pain was blocked by a cyclooxygenase-2 inhibitor, suggesting an indirect effect on neurons. Correspondingly, dorsal root ganglion neurons cultured in G-CSF failed to respond to G-CSF in vitro, and Csf3r gene expression could not be detected in dorsal root ganglion neurons by single-cell RT-PCR. These data suggest that G-CSFR/G-CSF targeting may be a safe therapeutic strategy for arthritis and other inflammatory conditions, particularly those in which pain is important, as well as for inflammatory pain per se.

Granulocyte macrophage colony-stimulating factor induces CCL17 production via IRF4 to mediate inflammation.

Data from preclinical and clinical studies have demonstrated that granulocyte macrophage colony-stimulating factor (GM-CSF) can function as a key proinflammatory cytokine. However, therapies that directly target GM-CSF function could lead to undesirable side effects, creating a need to delineate downstream pathways and mediators. In this work, we provide evidence that GM-CSF drives CCL17 production by acting through an IFN regulatory factor 4-dependent (IRF4-dependent) pathway in human monocytes, murine macrophages, and mice in vivo. In murine models of arthritis and pain, IRF4 regulated the formation of CCL17, which mediated the proinflammatory and algesic actions of GM-CSF. Mechanistically, GM-CSF upregulated IRF4 expression by enhancing JMJD3 demethylase activity. We also determined that CCL17 has chemokine-independent functions in inflammatory arthritis and pain. These findings indicate that GM-CSF can mediate inflammation and pain by regulating IRF4-induced CCL17 production, providing insights into a pathway with potential therapeutic avenues for the treatment of inflammatory diseases and their associated pain.

K/BxN Serum-Transfer Arthritis as a Model for Human Inflammatory Arthritis.

The K/BxN serum-transfer arthritis (STA) model is a murine model in which the immunological mechanisms occurring in rheumatoid arthritis (RA) and other arthritides can be studied. To induce K/BxN STA, serum from arthritic transgenic K/BxN mice is transferred to naive mice and manifestations of arthritis occur a few days later. The inflammatory response in the model is driven by autoantibodies against the ubiquitously expressed self-antigen, glucose-6-phosphate isomerase (G6PI), leading to the formation of immune complexes that drive the activation of different innate immune cells such as neutrophils, macrophages, and possibly mast cells. The pathogenesis further involves a range of immune mediators including cytokines, chemokines, complement factors, Toll-like receptors, Fc receptors, and integrins, as well as factors involved in pain and bone erosion. Hence, even though the K/BxN STA model mimics only the effector phase of RA, it still involves a wide range of relevant disease mediators. Additionally, as a murine model for arthritis, the K/BxN STA model has some obvious advantages. First, it has a rapid and robust onset of arthritis with 100% incidence in genetically identical animals. Second, it can be induced in a wide range of strain backgrounds and can therefore also be induced in gene-deficient strains to study the specific importance of disease mediators. Even though G6PI might not be an essential autoantigen, for example, in RA, the K/BxN STA model is a useful tool to understand how autoantibodies, in general, drive the progression of arthritis by interacting with downstream components of the innate immune system. Finally, the model has also proven useful as a model wherein arthritic pain can be studied. Taken together, these features make the K/BxN STA model a relevant one for RA, and it is a potentially valuable tool, especially for the preclinical screening of new therapeutic targets for RA and perhaps other forms of inflammatory arthritis. Here, we describe the molecular and cellular pathways in the development of K/BxN STA focusing on the recent advances in the understanding of the important mechanisms. Additionally, this review provides a comparison of the K/BxN STA model to some other arthritis models.

Granulocyte colony-stimulating factor (G-CSF) plays an important role in immune complex-mediated arthritis.

Neutrophils are an abundant cell type in many chronic inflammatory diseases such as rheumatoid arthritis (RA); however, their contribution to the pathology of RA has not been widely studied. A key cytokine involved in neutrophil development and function is granulocyte-colony stimulating factor (G-CSF). In this study we used the K/BxN serum-transfer arthritis (STA) model, mimicking the effector phase of RA, to investigate the importance of G-CSF in arthritis development and its relation to neutrophils. Here, we show for the first time in this model that G-CSF levels are increased both in the serum and in inflamed paws of arthritic mice and importantly that G-CSF blockade leads to a profound reduction in arthritis severity, as well as reduced numbers of neutrophils in blood. Moreover, CXCL1 and CXCL2 levels in the arthritic joints were also lowered. Our data demonstrate that G-CSF is a pivotal driver of the disease progression in the K/BxN STA model and possibly acts in part by regulating neutrophil numbers in the circulation. Therefore, our findings suggest that G-CSF might be a suitable target in RA, and perhaps in other immune complex-driven pathologies.