The DNA sensor AIM2 mediates psoriasiform inflammation by inducing type 3 immunity.

Psoriasis is a chronic and recurrent inflammatory skin disease characterized by abnormal proliferation and differentiation of keratinocytes and activation of immune cells. However, the molecular driver that triggers this immune response in psoriatic skin remains unclear. The inflammation-related gene absent in melanoma 2 (AIM2) was identified as a susceptibility gene/locus associated with psoriasis. In this study, we investigated the role of AIM2 in the pathophysiology of psoriasis. We found elevated levels of mitochondrial DNA in patients with psoriasis, along with high expression of AIM2 in both the human psoriatic epidermis and a mouse model of psoriasis induced by topical imiquimod (IMQ) application. Genetic ablation of AIM2 reduced the development of IMQ-induced psoriasis by decreasing the production of type 3 cytokines (such as IL-17A and IL-23) and infiltration of immune cells into the inflammatory site. Furthermore, we demonstrate that IL-17A induced AIM2 expression in keratinocytes. Finally, the genetic absence of inflammasome components downstream AIM2, ASC, and caspase-1 alleviated IMQ-induced skin inflammation. Collectively, our data show that AIM2 is involved in developing psoriasis through its canonical activation.

Mineralocorticoid receptor antagonism partially prevents dysfunction of T cell maturation in rats chronically treated with ethanol.

Ethanol consumption induces thymic atrophy and affects T cell maturation in the thymus. However, the mechanisms underlying such effects still need to be fully understood. We attempted to investigate the role of mineralocorticoid receptors (MR) on ethanol-induced thymic atrophy, T cell maturation dysfunction, and the role of oxidative stress in such responses. Male Wistar Hannover rats were treated with ethanol (20%; in volume ratio) and/or potassium canrenoate, an antagonist of MR (MRA; 30 mg/kg/day, gavage) for five weeks. Blockade of MR prevented ethanol-induced increases in the number of double-positive (CD4+CD8+), CD8+ single-positive (CD4-CD8+), CD4+ single-positive (CD4+CD8-), and Foxp3+CD4+ (Treg) cells in the thymus. Ethanol increased NOX2-derived superoxide (O2•-), lipoperoxidation, and superoxide dismutase (SOD) activity in the thymus. Pretreatment with the MRA fully prevented these responses. Apocynin, an antioxidant, prevented ethanol-induced increases in the number of double-positive and CD8+ single-positive cells but failed to prevent the rise in the number of CD4+ single-positive and Treg cells induced by ethanol. Apocynin, but not the MRA, prevented thymic atrophy induced by ethanol. Our findings provided novel evidence for the participation of MR in thymic dysfunction induced by ethanol consumption. Oxidative stress mediates the increase in double-positive and CD8+ single-positive cells in response to MR activation, while positive regulation of CD4+ single-positive and Treg cells is independent of oxidative stress. Oxidative stress is a significant mechanism of thymic atrophy associated with ethanol consumption, but this response is independent of MR activation.

Macrophage-derived human resistin promotes perivascular adipose tissue dysfunction in experimental inflammatory arthritis.

Cardiovascular disease (CVD) is the leading cause of death in rheumatoid arthritis (RA). Resistin is an adipokine that induces adipose tissue inflammation and activation of monocytes/macrophages via adenylate cyclase-associated protein-1 (CAP1). Resistin levels are increased in RA and might cause perivascular adipose tissue (PVAT) dysfunction, leading to vascular damage and CVD. This study aimed to investigate the role of resistin in promoting PVAT dysfunction by increasing local macrophage and inflammatory cytokines content in antigen-induced arthritis (AIA). Resistin pharmacological effects were assessed by using C57Bl/6J wild-type (WT) mice, humanized resistin mice expressing human resistin in monocytes-macrophages (hRTN+/-/-), and resistin knockout mice (RTN-/-) with AIA and respective controls. We investigated AIA disease activity and functional, cellular, and molecular parameters of the PVAT. Resistin did not contribute to AIA disease activity and its concentrations were augmented in the PVAT and plasma of WT AIA and hRTN+/-/- AIA animals. In vitro exposure of murine arteries to resistin impaired vascular function by decreasing the anti-contractile effect of PVAT. WT AIA mice and hRTN+/-/- AIA mice exhibited PVAT dysfunction and knockdown of resistin prevented it. Macrophage-derived cytokines, markers of types 1 and 2 macrophages, and CAP1 expression were increased in the PVAT of resistin humanized mice with AIA, but not in knockout mice for resistin. This study reveals that macrophage-derived resistin promotes PVAT inflammation and dysfunction regardless of AIA disease activity. Resistin might represent a translational target to reduce RA-driven vascular dysfunction and CVD.

Neuron-associated macrophage proliferation in the sensory ganglia is associated with peripheral nerve injury-induced neuropathic pain involving CX3CR1 signaling.

Resident macrophages are distributed across all tissues and are highly heterogeneous due to adaptation to different tissue-specific environments. The resident macrophages of the sensory ganglia (sensory neuron-associated macrophages, sNAMs) are in close contact with the cell body of primary sensory neurons and might play physiological and pathophysiological roles. After peripheral nerve injury, there is an increase in the population of macrophages in the sensory ganglia, which have been implicated in different conditions, including neuropathic pain development. However, it is still under debate whether macrophage accumulation in the sensory ganglia after peripheral nerve injury is due to the local proliferation of resident macrophages or a result of blood monocyte infiltration. Here, we confirmed that the number of macrophages increased in the sensory ganglia after the spared nerve injury (SNI) model in mice. Using different approaches, we found that the increase in the number of macrophages in the sensory ganglia after SNI is a consequence of the proliferation of resident CX3CR1+ macrophages, which participate in the development of neuropathic pain, but not due to infiltration of peripheral blood monocytes. These proliferating macrophages are the source of pro-inflammatory cytokines such as TNF and IL-1b. In addition, we found that CX3CR1 signaling is involved in the sNAMs proliferation and neuropathic pain development after peripheral nerve injury. In summary, these results indicated that peripheral nerve injury leads to sNAMs proliferation in the sensory ganglia in a CX3CR1-dependent manner accounting for neuropathic pain development. In conclusion, sNAMs proliferation could be modulated to change pathophysiological conditions such as chronic neuropathic pain.

Targeting neutrophils extracellular traps (NETs) reduces multiple organ injury in a COVID-19 mouse model.

BACKGROUND: COVID-19 is characterized by severe acute lung injury, which is associated with neutrophil infiltration and the release of neutrophil extracellular traps (NETs). COVID-19 treatment options are scarce. Previous work has shown an increase in NETs release in the lung and plasma of COVID-19 patients suggesting that drugs that prevent NETs formation or release could be potential therapeutic approaches for COVID-19 treatment. METHODS: Here, we report the efficacy of NET-degrading DNase I treatment in a murine model of COVID-19. SARS-CoV-2-infected K18-hACE2 mice were performed for clinical sickness scores and lung pathology. Moreover, the levels of NETs were assessed and lung injuries were by histopathology and TUNEL assay. Finally, the injury in the heart and kidney was assessed by histopathology and biochemical-specific markers. RESULTS: DNase I decreased detectable levels of NETs, improved clinical disease, and reduced lung, heart, and kidney injuries in SARS-CoV-2-infected K18-hACE2 mice. Furthermore, our findings indicate a potentially deleterious role for NETs lung tissue in vivo and lung epithelial (A549) cells in vitro, which might explain part of the pathophysiology of severe COVID-19. This deleterious effect was diminished by the treatment with DNase I. CONCLUSIONS: Together, our results support the role of NETs in COVID-19 immunopathology and highlight NETs disruption pharmacological approaches as a potential strategy to ameliorate COVID-19 clinical outcomes.

S100A9 Drives the Chronification of Psoriasiform Inflammation by Inducing IL-23/Type 3 Immunity.

Psoriasis is a chronic inflammatory skin disorder driven by the IL-23/type 3 immune response. However, molecular mechanisms sustaining the chronicity of inflammation and psoriatic lesions remain elusive. Combining systematic analyses of several transcriptomic datasets, we delineated gene signatures across human psoriatic skin, identifying S100A9 as one of the most up-regulated genes, which was confirmed in lesioned skin from patients with psoriasis and preclinical psoriasiform skin inflammation models. Genetic ablation or pharmacologic inhibition of S100A9 alleviated Aldara-induced skin inflammation. By single-cell mapping of human psoriatic skin and bone marrow chimeric mice experiments, we identified keratinocytes as the major source of S100A9. Mechanistically, S100A9 induced IL-23 production by dendritic cells, driving the IL-23/type 3 immunity in psoriasiform skin inflammation. In addition, the cutaneous IL-23/IL-17 axis induced epidermal S100A9 expression in human and experimental psoriasis. Thus, we showed an autoregulatory circuit between keratinocyte-derived S100A9 and IL-23/type 3 immunity during psoriasiform inflammation, identifying a crucial function of S100A9 in the chronification of psoriasis.

PD-1/PD-L1 Inhibition Enhances Chemotherapy-Induced Neuropathic Pain by Suppressing Neuroimmune Antinociceptive Signaling.

Cytotoxic agents synergize with immune checkpoint inhibitors and improve outcomes for patients with several cancer types. Nonetheless, a parallel increase in the incidence of dose-limiting side effects, such as peripheral neuropathy, is often observed. Here, we investigated the role of the programmed cell death-1 (PD-1)/programmed death-ligand 1 (PD-L1) axis in the modulation of paclitaxel-induced neuropathic pain. We found that human and mouse neural tissues, including the dorsal root ganglion (DRG), expressed basal levels of PD-1 and PD-L1. During the development of paclitaxel-induced neuropathy, an increase in PD-L1 expression was observed in macrophages from the DRG. This effect depended on Toll-like receptor 4 activation by paclitaxel. Furthermore, PD-L1 inhibited pain behavior triggered by paclitaxel or formalin in mice, suggesting that PD-1/PD-L1 signaling attenuates peripheral neuropathy development. Consistent with this, we observed that the combined use of anti-PD-L1 plus paclitaxel increased mechanical allodynia and chronic neuropathy development induced by single agents. This effect was associated with higher expression of inflammatory markers (Tnf, Il6, and Cx3cr1) in peripheral nervous tissue. Together, these results suggest that PD-1/PD-L1 inhibitors enhance paclitaxel-induced neuropathic pain by suppressing PD-1/PD-L1 antinociceptive signaling.

Efferocytosis of SARS-CoV-2-infected dying cells impairs macrophage anti-inflammatory functions and clearance of apoptotic cells.

COVID-19 is a disease of dysfunctional immune responses, but the mechanisms triggering immunopathogenesis are not established. The functional plasticity of macrophages allows this cell type to promote pathogen elimination and inflammation or suppress inflammation and promote tissue remodeling and injury repair. During an infection, the clearance of dead and dying cells, a process named efferocytosis, can modulate the interplay between these contrasting functions. Here, we show that engulfment of SARS-CoV-2-infected apoptotic cells exacerbates inflammatory cytokine production, inhibits the expression of efferocytic receptors, and impairs continual efferocytosis by macrophages. We also provide evidence supporting that lung monocytes and macrophages from severe COVID-19 patients have compromised efferocytic capacity. Our findings reveal that dysfunctional efferocytosis of SARS-CoV-2-infected cell corpses suppresses macrophage anti-inflammation and efficient tissue repair programs and provides mechanistic insights for the excessive production of pro-inflammatory cytokines and accumulation of tissue damage associated with COVID-19 immunopathogenesis.

Th17 cell-linked mechanisms mediate vascular dysfunction induced by testosterone in a mouse model of gender-affirming hormone therapy.

Clinical data point to adverse cardiovascular events elicited by testosterone replacement therapy. Testosterone is the main hormone used in gender-affirming hormone therapy (GAHT) by transmasculine people. However, the cardiovascular impact of testosterone in experimental models of GAHT remains unknown. Sex hormones modulate T-cell activation, and immune mechanisms contribute to cardiovascular risk. The present study evaluated whether testosterone negatively impacts female cardiovascular function by enhancing Th17 cell-linked effector mechanisms. Female (8 wk old) C57BL/6J mice received testosterone (48 mg/kg/wk) for 8 wk. Male mice were used for phenotypical comparisons. The hormone treatment in female mice increased circulating testosterone to levels observed in male mice. Testosterone increased lean body mass and body mass index, and decreased perigonadal fat mass, mimicking clinical findings. After 8 wk, testosterone decreased endothelium-dependent vasodilation and increased peripheral Th17 cells. After 24 wk, testosterone increased blood pressure in female mice. Ovariectomy did not intensify phenotypical or cardiovascular effects by testosterone. Female mice lacking T and B cells [Rag1 knockout (-/-)], as well as female mice lacking IL-17 receptor (IL-17Ra-/-), did not exhibit vascular dysfunction induced by testosterone. Testosterone impaired endothelium-dependent vasodilation in female mice lacking γδ T cells, similarly to the observed in wild-type female mice. Adoptive transfer of CD4+ T cells restored testosterone-induced vascular dysfunction in Rag1-/- female mice. Together, these data suggest that CD4+ T cells, most likely Th17 cells, are central to vascular dysfunction induced by testosterone in female mice, indicating that changes in immune-cell balance are important in the GAHT in transmasculine people.NEW & NOTEWORTHY Sex hormone-induced cardiovascular events are important undesirable effects in transgender people under GAHT. Studies addressing the cardiovascular impact of GAHT will certainly contribute to improve healthcare services offered to this population. Our study showing that vascular dysfunction, via Th17 cell-related mechanisms, precedes increased blood pressure induced by testosterone in a GAHT mouse model, reveals potential mechanisms involved in GAHT-related cardiovascular events and may provide new markers/targets for clinical practices in transmasculine people.

SARS-CoV-2 productively infects primary human immune system cells in vitro and in COVID-19 patients.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is associated with a hyperinflammatory state and lymphocytopenia, a hallmark that appears as both signature and prognosis of disease severity outcome. Although cytokine storm and a sustained inflammatory state are commonly associated with immune cell depletion, it is still unclear whether direct SARS-CoV-2 infection of immune cells could also play a role in this scenario by harboring viral replication. We found that monocytes, as well as both B and T lymphocytes, were susceptible to SARS-CoV-2 infection in vitro, accumulating double-stranded RNA consistent with viral RNA replication and ultimately leading to expressive T cell apoptosis. In addition, flow cytometry and immunofluorescence analysis revealed that SARS-CoV-2 was frequently detected in monocytes and B lymphocytes from coronavirus disease 2019 (COVID-19) patients. The rates of SARS-CoV-2-infected monocytes in peripheral blood mononuclear cells from COVID-19 patients increased over time from symptom onset, with SARS-CoV-2-positive monocytes, B cells, and CD4+ T lymphocytes also detected in postmortem lung tissue. These results indicated that SARS-CoV-2 infection of blood-circulating leukocytes in COVID-19 patients might have important implications for disease pathogenesis and progression, immune dysfunction, and virus spread within the host.

The Combination of Gefitinib With ATRA and ATO Induces Myeloid Differentiation in Acute Promyelocytic Leukemia Resistant Cells.

In approximately 15% of patients with acute myeloid leukemia (AML), total and phosphorylated EGFR proteins have been reported to be increased compared to healthy CD34+ samples. However, it is unclear if this subset of patients would benefit from EGFR signaling pharmacological inhibition. Pre-clinical studies on AML cells provided evidence on the pro-differentiation benefits of EGFR inhibitors when combined with ATRA or ATO in vitro. Despite the success of ATRA and ATO in the treatment of patients with acute promyelocytic leukemia (APL), therapy-associated resistance is observed in 5-10% of the cases, pointing to a clear need for new therapeutic strategies for those patients. In this context, the functional role of EGFR tyrosine-kinase inhibitors has never been evaluated in APL. Here, we investigated the EGFR pathway in primary samples along with functional in vitro and in vivo studies using several APL models. We observed that total and phosphorylated EGFR (Tyr992) was expressed in 28% and 19% of blast cells from APL patients, respectively, but not in healthy CD34+ samples. Interestingly, the expression of the EGF was lower in APL plasma samples than in healthy controls. The EGFR ligand AREG was detected in 29% of APL patients at diagnosis, but not in control samples. In vitro, treatment with the EGFR inhibitor gefitinib (ZD1839) reduced cell proliferation and survival of NB4 (ATRA-sensitive) and NB4-R2 (ATRA-resistant) cells. Moreover, the combination of gefitinib with ATRA and ATO promoted myeloid cell differentiation in ATRA- and ATO-resistant APL cells. In vivo, the combination of gefitinib and ATRA prolonged survival compared to gefitinib- or vehicle-treated leukemic mice in a syngeneic transplantation model, while the gain in survival did not reach statistical difference compared to treatment with ATRA alone. Our results suggest that gefitinib is a potential adjuvant agent that can mitigate ATRA and ATO resistance in APL cells. Therefore, our data indicate that repurposing FDA-approved tyrosine-kinase inhibitors could provide new perspectives into combination therapy to overcome drug resistance in APL patients.

Beneficial effects of colchicine for moderate to severe COVID-19: a randomised, double-blinded, placebo-controlled clinical trial.

OBJECTIVE: To evaluate whether the addition of colchicine to standard treatment for COVID-19 results in better outcomes. DESIGN: We present the results of a randomised, double-blinded, placebo-controlled clinical trial of colchicine for the treatment of moderate to severe COVID-19, with 75 patients allocated 1:1 from 11 April to 30 August 2020. Colchicine regimen was 0.5 mg thrice daily for 5 days, then 0.5 mg twice daily for 5 days. The primary endpoints were the need for supplemental oxygen, time of hospitalisation, need for admission and length of stay in intensive care unit and death rate. RESULTS: Seventy-two patients (36 for placebo and 36 for colchicine) completed the study. Median (and IQR) time of need for supplemental oxygen was 4.0 (2.0-6.0) days for the colchicine group and 6.5 (4.0-9.0) days for the placebo group (p<0.001). Median (IQR) time of hospitalisation was 7.0 (5.0-9.0) days for the colchicine group and 9.0 (7.0-12.0) days for the placebo group (p=0.003). At day 2, 67% versus 86% of patients maintained the need for supplemental oxygen, while at day 7, the values were 9% versus 42%, in the colchicine and the placebo groups, respectively (log rank; p=0.001). Two patients died, both in placebo group. Diarrhoea was more frequent in the colchicine group (p=0.26). CONCLUSION: Colchicine reduced the length of both, supplemental oxygen therapy and hospitalisation. The drug was safe and well tolerated. Once death was an uncommon event, it is not possible to ensure that colchicine reduced mortality of COVID-19. TRIAL REGISTRATION NUMBER: RBR-8jyhxh.

The PI3Kγ/AKT signaling pathway mediates peripheral antinociceptive action of dipyrone.

Dipyrone (DIP), also known as metamizole, is an over-the-counter analgesic used in Europe and Latin America. Evidence suggesting that inflammatory pain attenuation by DIP is associated with a direct impact on peripheral primary nociceptive neurons through the stimulation of nitric oxide signaling pathway. However, the molecular mechanism by which DIP activates this pathway remains unknown. The PI3Kγ/AKT signaling cascade activation is one of the well-known molecular mechanisms that promote nitric oxide production in sensory neurons. Herein, we investigated the role of the PI3Kγ/AKT signaling cascade in the context of peripheral analgesic effect of DIP. DIP was administered into PGE2 pre-sensitized paws of rats and mechanical hyperalgesia was determined using electronic von Frey test after 1 h. Nonselective or selective pharmacological inhibitors of PI3Kγ and AKT were also administered in DIP-treated rats under paws sensitized with PGE2. Intraplantar injection of DIP attenuated PGE2-induced hyperalgesia in a dose-dependent manner. Treatment with nonselective (wortmannin or LY294002) or selective (AS605240) pharmacological inhibitors of PI3Kγ reduced the peripheral antihypernociceptive effect of DIP. Consistently, AKT selective inhibitor also reversed analgesic DIP effects. Corroborating these data, we found that DIP induced AKT phosphorylation in cultured dorsal root ganglion neurons, which was prevented in the presence of PI3Kγ selective inhibitor. Taken together, these findings provide evidence that peripheral analgesic effect of DIP is dependent on the activation of PI3Kγ/AKT signaling pathway.

Inflammasomes are activated in response to SARS-CoV-2 infection and are associated with COVID-19 severity in patients.

Severe cases of COVID-19 are characterized by a strong inflammatory process that may ultimately lead to organ failure and patient death. The NLRP3 inflammasome is a molecular platform that promotes inflammation via cleavage and activation of key inflammatory molecules including active caspase-1 (Casp1p20), IL-1ß, and IL-18. Although participation of the inflammasome in COVID-19 has been highly speculated, the inflammasome activation and participation in the outcome of the disease are unknown. Here we demonstrate that the NLRP3 inflammasome is activated in response to SARS-CoV-2 infection and is active in COVID-19 patients. Studying moderate and severe COVID-19 patients, we found active NLRP3 inflammasome in PBMCs and tissues of postmortem patients upon autopsy. Inflammasome-derived products such as Casp1p20 and IL-18 in the sera correlated with the markers of COVID-19 severity, including IL-6 and LDH. Moreover, higher levels of IL-18 and Casp1p20 are associated with disease severity and poor clinical outcome. Our results suggest that inflammasomes participate in the pathophysiology of the disease, indicating that these platforms might be a marker of disease severity and a potential therapeutic target for COVID-19.

Molecular basis of carrageenan-induced cytokines production in macrophages.

BACKGROUND: Low molecular weight carrageenan (Cg) is a seaweed-derived sulfated polysaccharide widely used as inflammatory stimulus in preclinical studies. However, the molecular mechanisms of Cg-induced inflammation are not fully elucidated. The present study aimed to investigate the molecular basis involved in Cg-induced macrophages activation and cytokines production. METHODS: Primary culture of mouse peritoneal macrophages were stimulated with Kappa Cg. The supernatant and cell lysate were used for ELISA, western blotting, immunofluorescence. Cg-induced mouse colitis was also developed. RESULTS: Here we show that Cg activates peritoneal macrophages to produce pro-inflammatory cytokines such as TNF and IL-1ß. While Cg-induced TNF production/secretion depends on TLR4/MyD88 signaling, the production of pro-IL-1ß relies on TLR4/TRIF/SYK/reactive oxygen species (ROS) signaling pathway. The maturation of pro-IL1ß into IL-1ß is dependent on canonical NLRP3 inflammasome activation via Pannexin-1/P2X7/K+ efflux signaling. In vivo, Cg-induced colitis was reduced in mice in the absence of NLRP3 inflammasome components. CONCLUSIONS: In conclusion, we unravel a critical role of the NLRP3 inflammasome in Cg-induced pro-inflammatory cytokines production and colitis, which is an important discovery on the pro-inflammatory properties of this sulfated polysaccharide for pre-clinical studies. Video abstract Carrageenan (Cg) is one the most used flogistic stimulus in preclinical studies. Nevertheless, the molecular basis of Cg-induced inflammation is not totally elucidated. Herein, Lopes et al. unraveled the molecular basis for Cg-induced macrophages production of biological active IL-1ß. The Cg-stimulated macrophages produces pro-IL-1ß depends on TLR4/TRIF/Syk/ROS, whereas its processing into mature IL-1ß is dependent on the canonical NLRP3 inflammasome.

IL-33 enhances macrophage release of IL-1ß and promotes pain and inflammation in gouty arthritis.

OBJECTIVE: To investigate the role of IL-33 in gouty arthritis. MATERIAL: 174 Balb/c (wild-type) and 54 ST2-/- mice were used in this study. In vitro experiments were conducted in bone marrow-derived macrophages (BMDMs). Synovial fluid samples from gouty arthritis (n = 7) and osteoarthritis (n = 8) hospital patients were used to measure IL-33 and sST2 levels. METHODS: Gout was induced by injection of monosodium urate (MSU) crystals in the knee joint of mice. Pain was determined using the electronic von Frey and static weight bearing. Neutrophil recruitment was determined by H&E staining, Rosenfeld staining slides, and MPO activity. ELISA was used for cytokine and sST2 measurement. The priming effect of IL-33 was determined in BMDM. RESULTS: Synovial fluid of gout patients showed higher IL-33 levels and neutrophil counts than osteoarthritis patients. In mice, the absence of ST2 prevented mechanical pain, knee joint edema, neutrophil recruitment to the knee joint, and lowered IL-1ß and superoxide anion levels. In macrophages, IL-33 enhanced the release of IL-1ß and TNF-α, and BMDMs from ST2-/- showed reduced levels of these cytokines after stimulus with MSU crystals. CONCLUSION: IL-33 mediates gout pain and inflammation by boosting macrophages production of cytokines upon MSU crystals stimulus.

MEK5/ERK5 signaling mediates IL-4-induced M2 macrophage differentiation through regulation of c-Myc expression.

Macrophages are highly plastic cells, responding to diverse environmental stimuli to acquire different functional phenotypes. Signaling through MAPKs has been reported to regulate the differentiation of macrophages, but the role of ERK5 in IL-4-mediated M2 macrophage differentiation is still unclear. Here, we showed that the ERK5 signaling pathway plays a critical role in IL-4-induced M2 macrophage differentiation. Pharmacologic inhibition of MEK5, an upstream activator of ERK5, markedly reduced the expression of classical M2 markers, such as Arg-1, Ym-1, and Fizz-1, as well as the production of M2-related chemokines and cytokines, CCL22, CCL17, and IGF-1 in IL-4-stimulated macrophages. Moreover, pharmacologic inhibition of ERK5 also decreased the expression of several M2 markers induced by IL-4. In accordance, myeloid cell-specific Erk5 depletion (Erk5∆mye ), using LysMcre /Erk5f/f mice, confirmed the involvement of ERK5 in IL-4-induced M2 polarization. Mechanistically, the inhibition of ERK5 did not affect STAT3 or STAT6 phosphorylation, suggesting that ERK5 signaling regulates M2 differentiation in a STAT3 and STAT6-independent manner. However, genetic deficiency or pharmacologic inhibition of the MEK5/ERK5 pathway reduced the expression of c-Myc in IL-4-activated macrophages, which is a critical transcription factor involved in M2 differentiation. Our study thus suggests that the MEK5/ERK5 signaling pathway is crucial in IL-4-induced M2 macrophage differentiation through the induction of c-Myc expression.

Regulatory T cells counteract neuropathic pain through inhibition of the Th1 response at the site of peripheral nerve injury.

The inflammatory/immune response at the site of peripheral nerve injury participates in the pathophysiology of neuropathic pain. Nevertheless, little is known about the local regulatory mechanisms underlying peripheral nerve injury that counteracts the development of pain. Here, we investigated the contribution of regulatory T (Treg) cells to the development of neuropathic pain by using a partial sciatic nerve ligation model in mice. We showed that Treg cells infiltrate and proliferate in the site of peripheral nerve injury. Local Treg cells suppressed the development of neuropathic pain mainly through the inhibition of the CD4 Th1 response. Treg cells also indirectly reduced neuronal damage and neuroinflammation at the level of the sensory ganglia. Finally, we identified IL-10 signaling as an intrinsic mechanism by which Treg cells counteract neuropathic pain development. These results revealed Treg cells as important inhibitory modulators of the immune response at the site of peripheral nerve injury that restrains the development of neuropathic pain. In conclusion, the boosting of Treg cell function/activity might be explored as a possible interventional approach to reduce neuropathic pain development after peripheral nerve damage.

IL-22 Promotes IFN-γ-Mediated Immunity against Histoplasma capsulatum Infection.

Histoplasma capsulatum is the agent of histoplasmosis, one of the most frequent mycoses in the world. The infection initiates with fungal spore inhalation, transformation into yeasts in the lungs and establishment of a granulomatous disease, which is characterized by a Th1 response. The production of Th1 signature cytokines, such as IFN-γ, is crucial for yeast clearance from the lungs, and to prevent dissemination. Recently, it was demonstrated that IL-17, a Th17 signature cytokine, is also important for fungal control, particularly in the absence of Th1 response. IL-22 is another cytokine with multiple functions on host response and disease progression. However, little is known about the role of IL-22 during histoplasmosis. In this study, we demonstrated that absence of IL-22 affected the clearance of yeasts from the lungs and increased the spreading to the spleen. In addition, IL-22 deficient mice (Il22-/-) succumbed to infection, which correlated with reductions in the numbers of CD4+ IFN-γ+ T cells, reduced IFN-γ levels, and diminished nitric oxide synthase type 2 (NOS2) expression in the lungs. Importantly, treatment with rIFN-γ mitigated the susceptibility of Il22-/- mice to H. capsulatum infection. These data indicate that IL-22 is crucial for IFN-γ/NO production and resistance to experimental histoplasmosis.

Frontline Science: Blood-circulating leukocytes fail to infiltrate the spinal cord parenchyma after spared nerve injury.

The development of neuropathic pain after peripheral nerve injury involves neuroimmune-glial interactions in the spinal cord. However, whether the development of neuropathic pain depends on the infiltration of peripheral immune cells, such as monocytes, into the spinal cord parenchyma after peripheral nerve damage remains unclear. Here, we used a combination of different techniques such as transgenic reporter mouse (Cx3cr1GFP/+ and Ccr2RFP/+ mice), bone marrow chimeric mice, and parabiosis to investigate this issue in spared nerve injury (SNI) model. Herein, we provided robust evidence that, although microglial cells are activated/proliferate at the dorsal horn of the spinal cord after SNI, peripheral hematopoietic cells (including monocytes) are not able to infiltrate into the spinal cord parenchyma. Furthermore, there was no evidence of CCR2 expression in intrinsic cells of the spinal cord. However, microglial cells activation/proliferation in the spinal cord and mechanical allodynia after SNI were reduced in Ccr2-deficient mice. These results suggest that blood-circulating leukocytes cells are not able to infiltrate the spinal cord parenchyma after distal peripheral nerve injury. Nevertheless, they indicate that CCR2-expressing cells might be indirectly regulating microglia activation/proliferation in the spinal cord after SNI. In conclusion, our study supports that CCR2 inhibition could be explored as an interventional approach to reduce microglia activation and consequently neuropathic pain development after peripheral nerve injury.