Influx Of Monocytes Research Articles

Role of Jak3 in hepatic inflammation during ulcerative colitis.

BackgroundJanus kinase 3 (Jak3) is a non‐receptor protein kinase that plays essential role in hematopoietic and intestinal epithelial differentiation. Previously, we reported that loss of Jak3 exacerbates symptoms in murine model of DSS‐induced colitis through compromised intestinal differentiation of goblet cells. Studies suggest a linkage between colitis and hepatic steatosis, but the genetic regulation of such a regulation and the role of Jak3 in hepatic steatosis is not known. In this work, we investigated the tissue‐specific role of Jak3 in maintaining hepatic homeostasis during intestinal inflammation.MethodsWe used a global Jak3 KO (Jak3−/−) mice and their littermate control wild type mice in a model of DSS‐induced colitis and compared the effects of Jak3 on hepatic steatosis and the status of hepatic TLR signaling and TGFb1 pathways. We also investigated the tissue specific roles of Jak3 in liver steatosis during DSS‐induced colitis using organs collected at the end of the study and analyzed using a combination of flow cytometry, confocal microscopy, and western analysis techniques.ResultsOur results show increased severity towards DSS‐induced colitis in Jak3−/− mice, IEC‐Jak3−/− and IMM‐Jak3−/−compared to their littermates controls. The inflammations in Jak3−/− or IEC‐Jak3−/− and IMM‐Jak3−/−mice showed increased disease activity scores that were associated with shortening of colon length, reduced cecum length, decreased crypt heights, increased liver to body weight ratio, and increased hepatic steatosis compared to their littermate controls. To determine the tissue specific role of Jak3, our data show a high‐degree of association between ulcerative colitis and hepatic inflammation in all three Jak3‐KO groups indicating an essential role of Jak3 in maintaining tissue homeostasis and immunotolerance. IHC studies indicated a role of Kupffer cells in hepatic inflammation. A differential flowcytometric assay showed an increased influx of monocytes, but reduced differentiation of residential Kupffer cells in Jak3 KO mice compared to control. Moreover, KO mice showed an increased hepatic expression of TLR‐2 and TLR‐4 and a higher IL‐6, IL‐17 and Tnf‐α as compared to the control mice. Genetic analysis showed, either intestinal inflammation by DSS or loss of Jak3 without DSS both resulted in increased expression of TGFb1, Fn1, and decreased expression of Cola1 and aSMA. Together, these results showed that loss of Jak3 expression leads to compromise tolerance of Kupffer cells coupled with increased extravasation of the circulating monocytes into the liver. This could lead to exacerbation of hepatic inflammation in ulcerative colitis. In addition, we also found an increased Th17 effector function and simultaneous suppression of Treg cell proliferation in the absence of Jak3, when compared to its control mice.ConclusionCollectively, these results indicate that Jak3 plays a major role in regulating the hepatic inflammation during ulcerative colitis.

High-salt intake increases TRPC3 expression and enhances TRPC3-mediated calcium influx and systolic blood pressure in hypertensive patients.

Enhanced transient receptor potential canonical subtype 3 (TRPC3) expression and TRPC3-mediated calcium influx in monocytes from hypertensive rats and patients are associated with increased blood pressure. Daily salt intake is closely related to hypertension, but the relationship between TRPC3 expression and salt intake has not yet been evaluated in hypertensive patients. Using reverse transcription-polymerase chain reaction, we studied the expression of TRPC3 and TRPC3-related store-operated calcium entry (SOCE) in peripheral blood mononuclear cells (PBMCs) from hypertensive and normotensive control subjects. Measurement of SOCE was performed using the fluorescent dye Fura-2 AM. Participants were divided into a low-salt group (<9 g) and a high-salt group (≥9 g) based on 24-h urinary sodium excretion. Increased TRPC3 mRNA expression levels and SOCE were observed in THP-1 cells after high-NaCl treatment. However, administration of the TRPC3-specific inhibitor Pyr3 significantly decreased the effect. Furthermore, the TRPC3 mRNA expression levels in PBMCs from high-salt intake patients with essential hypertension were significantly higher than those in low-salt intake patients compared with those in normotensive control subjects. We also observed significantly increased TRPC3-mediated SOCE in PBMCs from hypertensive subjects (but not from normotensive control subjects), with calcium concentration correlating with salt intake. More importantly, TRPC3 mRNA levels showed a significant correlation with salt intake and systolic blood pressure in patients with essential hypertension. This study demonstrated, for the first time, that increased TRPC3 mRNA levels are associated with elevated salt intake and systolic blood pressure in hypertensive patients.

MyD88-dependent influx of monocytes and neutrophils impairs lymph node B cell responses to chikungunya virus infection via Irf5, Nos2 and Nox2.

Humoral immune responses initiate in the lymph node draining the site of viral infection (dLN). Some viruses subvert LN B cell activation; however, our knowledge of viral hindrance of B cell responses of important human pathogens is lacking. Here, we define mechanisms whereby chikungunya virus (CHIKV), a mosquito-transmitted RNA virus that causes outbreaks of acute and chronic arthritis in humans, hinders dLN antiviral B cell responses. Infection of WT mice with pathogenic, but not acutely cleared CHIKV, induced MyD88-dependent recruitment of monocytes and neutrophils to the dLN. Blocking this influx improved lymphocyte accumulation, dLN organization, and CHIKV-specific B cell responses. Both inducible nitric oxide synthase (iNOS) and the phagocyte NADPH oxidase (Nox2) contributed to impaired dLN organization and function. Infiltrating monocytes expressed iNOS through a local IRF5- and IFNAR1-dependent pathway that was partially TLR7-dependent. Together, our data suggest that pathogenic CHIKV triggers the influx and activation of monocytes and neutrophils in the dLN that impairs virus-specific B cell responses.

Combination of sivelestat and N-acetylcysteine alleviates the inflammatory response and exceeds standard treatment for acetaminophen-induced liver injury.

Hepatocyte death during acetaminophen (APAP) intoxication elicits a reactive inflammatory response, with hepatic recruitment of neutrophils and monocytes, which further aggravates liver injury. Neutrophil elastase (NE), secreted by activated neutrophils, carries degradative and cytotoxic functions and maintains a proinflammatory state. We investigated NE as a therapeutic target in acetaminophen-induced liver injury (AILI). C57BL/6 mice were administered a toxic dose of APAP, 2h prior to receiving the NE inhibitor sivelestat, N-acetylcysteine (NAC), or a combination therapy, and were euthanized after 24 and 48h. Upon APAP overdose, neutrophils and monocytes infiltrate the injured liver, accompanied by increased levels of NE. Combination therapy of NAC and sivelestat significantly limits liver damage, as evidenced by lower serum transaminase levels and less hepatic necrosis compared to mice that received APAP only, and this to a greater extent than NAC monotherapy. Lower hepatic expression of proinflammatory markers was observed in the combination treatment group, and flow cytometry revealed significantly less monocyte influx in livers from mice treated with the combination therapy, compared to untreated mice and mice treated with NAC only. The potential of NE to induce leukocyte migration was confirmed in vitro. Importantly, sivelestat did not impair hepatic repair. In conclusion, combination of NE inhibition with sivelestat and NAC dampens the inflammatory response and reduces liver damage following APAP overdose. This strategy exceeds the standard of care and might represent a novel therapeutic option for AILI.

SINUSOIDAL CELLS AND CYTOKINE RESPONSE IN THE TETRACHLOROMETHANE-INDUCED HEPATOTOXICITY AND AN APPROACH TO ITS CORRECTION

High occurence of liver diseases (toxic, viral hepatitis, liver failure, cirrhosis) requires urgent search of new methods for management of the hepatobiliary diseases. At the present time, the role of immune mechanisms in pathogenesis of diffuse toxic liver damage is not finally clarified. The model of toxic hepatitis induced by carbon tetrachloride (CCl4) is widely known, but this approach allows us to perform complex evaluation and develop the methods for adequate correction of liver disorders in experimental model, which is not always feasible in clinical setting.To design a model of diffuse toxic liver damage, the CCl4 oil solution was used, having been administered intraperitoneally to experimental animals, at a single dose of 50 mg per 100 g body mass. Aiming for correction of toxic liver damage, the injections of aminophthalhydrazide (APH) to experimental animals were carried out intramuscularly at the dose of 2 mg/kg over the terms of experiment. An evaluation of the role of sinusoidal cells (SC) and cytokine production at the local and systemic level were carried out in the model of toxic liver damage caused by CCl4 and its correction by APH treatment. In the course of developing diffuse toxic liver damage induced by CCl4, the production of proinflammatory cytokines TNFα, IL-1α and IL-18 was enhanced at the local level, whereas an increase in TNFα concentration was observed in blood plasma. Following aminophthalhydrazide (APH) administration, the concentrations of proinflammatory cytokines (TNFα and IL-18) decreased at system level, along with locally decreased levels of IL-6 and IFNγ.Changes in the functional state of immunocompetent cells, which include sinusoidal cells (SC), have a significant impact on the development of pathological processes in the liver. The results of our study presume that, over the early periods of toxic impact upon liver tissue, the number of SCs increases both due to influx of blood monocytes and mature macrophages from the peritoneal cavity that enter the injury site directly via mesothelial layer. The SCs provide phagocytosis of damaged hepatocytes and contribute to resolution of the inflammatory process.Modulation of the macrophage activities by APH contributes to increased amounts of SCs at the early stages, and stabilizes their quantities after 2 weeks of APH injections. Change in the numbers of liver SCs during toxic damage affects the production of cytokines. A direct effect of APH upon the SCs may change the production of regulatory factors and compensate the insufficient rate of recovery processes after the toxic damage.

Caspase-11 Mediates Neutrophil Chemotaxis and Extracellular Trap Formation During Acute Gouty Arthritis Through Alteration of Cofilin Phosphorylation.

Gout is characterized by attacks of arthritis with hyperuricemia and monosodium urate (MSU) crystal-induced inflammation within joints. Innate immune responses are the primary drivers for tissue destruction and inflammation in gout. MSU crystals engage the Nlrp3 inflammasome, leading to the activation of caspase-1 and production of IL-1β and IL-18 within gout-affected joints, promoting the influx of neutrophils and monocytes. Here, we show that caspase-11−/− mice and their derived macrophages produce significantly reduced levels of gout-specific cytokines including IL-1β, TNFα, IL-6, and KC, while others like IFNγ and IL-12p70 are not altered. IL-1β induces the expression of caspase-11 in an IL-1 receptor-dependent manner in macrophages contributing to the priming of macrophages during sterile inflammation. The absence of caspase-11 reduced the ability of macrophages and neutrophils to migrate in response to exogenously injected KC in vivo. Notably, in vitro, caspase-11−/− neutrophils displayed random migration in response to a KC gradient when compared to their WT counterparts. This phenotype was associated with altered cofilin phosphorylation. Unlike their wild-type counterparts, caspase-11−/− neutrophils also failed to produce neutrophil extracellular traps (NETs) when treated with MSU. Together, this is the first report demonstrating that caspase-11 promotes neutrophil directional trafficking and function in an acute model of gout. Caspase-11 also governs the production of inflammasome-dependent and -independent cytokines from macrophages. Our results offer new, previously unrecognized functions for caspase-11 in macrophages and neutrophils that may apply to other neutrophil-mediated disease conditions besides gout.

Flow Cytometry Characterization of Cerebrospinal Fluid Monocytes in Patients With Postoperative Cognitive Dysfunction: A Pilot Study.

Animal models suggest postoperative cognitive dysfunction may be caused by brain monocyte influx. To study this in humans, we developed a flow cytometry panel to profile cerebrospinal fluid (CSF) samples collected before and after major noncardiac surgery in 5 patients ≥60 years of age who developed postoperative cognitive dysfunction and 5 matched controls who did not. We detected 12,654 ± 4895 cells/10 mL of CSF sample (mean ± SD). Patients who developed postoperative cognitive dysfunction showed an increased CSF monocyte/lymphocyte ratio and monocyte chemoattractant protein 1 receptor downregulation on CSF monocytes 24 hours after surgery. These pilot data demonstrate that CSF flow cytometry can be used to study mechanisms of postoperative neurocognitive dysfunction.

P6303Developmental origin of cardiac macrophages in steady state and myocardial infarction

Abstract Background Macrophages are the most abundant immune cells in the myocardial tissue in steady state. The sterile inflammation caused by myocardial infarction triggers a massive immune reaction, which leads to a profound influx of neutrophils and monocytes. In the postacute phase of infarction macrophages play an essential role in reparative processes. Recently, it has become clear that macrophages in the heart have a dual developmental origin from embryonic and bone marrow (BM) hematopoiesis. In this study, we sought to investigate the contribution of embryonic derived macrophages to the cardiac macrophage pool in steady state as well as the acute and chronic phase after ischemia/reperfusion injury. Methods/Results To address the origin of macrophages in steady state we used different models of lineage tracing to determine the developmental origin of cardiac macrophages. Using FLT3-Cre mice and radiation-independent CD45.1/.2 bone marrow chimera, we found that the resident macrophage population in the heart is mainly independent of definitive hematopoiesis (approximately 70–80% of cardiac macrophages). The BM-dependent population on the other hand is replenished by blood-derived monocytes. Further we used the radiation-independent CD45.1/.2 bone marrow chimera to characterize the origin of macrophages at different time points after I/R-injury. In the acute phase after myocardial infarction we observed a profound influx of BM-derived macrophages in the infarct region and also in the remote area. 30 days after I/R-injury the composition of the resident macrophage pool was mainly comprised of BM-independent macrophages, similar to steady state conditions. To address the role of BM-derived macrophages we used CCR2-ko mice, which have low numbers of inflammatory monocytes in peripheral blood. CCR2-ko mice showed reduced macrophage numbers in the acute phase after myocardial infarction. Using positron emission tomography we investigated the influence of CCR2-deficiency on cardiac function after I/R-injury. In comparison to WT mice, CCR2-ko mice showed a significantly increased infarct size. Cardiac remodeling, determined by end-diastolic volume, on the other hand was improved in CCR2-ko mice. The ejection fraction was similar in both groups. Conclusion The cardiac macrophage pool is mainly comprised of BM-independent macrophages. In response to I/R-injury monocyte-derived macrophages transiently enter the myocardium but do not persist in significant numbers over time. The influx of BM-derived macrophages after I/R-injury was reduced using CCR2-ko mice, which led to improved cardiac remodeling. Our findings are of potential importance for understanding the cardiac immune response and for the therapeutic targeting of macrophages in inflammatory conditions. Acknowledgement/Funding German Society of Cardiology, German Centre for Cardiovascular Research, LMU Excellence, SFB 914

Myelin status and oligodendrocyte lineage cells over time after spinal cord injury: What do we know and what still needs to be unwrapped?

Spinal cord injury (SCI) affects over 17,000 individuals in the United States per year, resulting in sudden motor, sensory and autonomic impairments below the level of injury. These deficits may be due at least in part to the loss of oligodendrocytes and demyelination of spared axons as it leads to slowed or blocked conduction through the lesion site. It has long been accepted that progenitor cells form new oligodendrocytes after SCI, resulting in the acute formation of new myelin on demyelinated axons. However, the chronicity of demyelination and the functional significance of remyelination remain contentious. Here we review work examining demyelination and remyelination after SCI as well as the current understanding of oligodendrocyte lineage cell responses to spinal trauma, including the surprisingly long-lasting response of NG2+ oligodendrocyte progenitor cells (OPCs) to proliferate and differentiate into new myelinating oligodendrocytes for months after SCI. OPCs are highly sensitive to microenvironmental changes, and therefore respond to the ever-changing post-SCI milieu, including influx of blood, monocytes and neutrophils; activation of microglia and macrophages; changes in cytokines, chemokines and growth factors such as ciliary neurotrophic factor and fibroblast growth factor-2; glutamate excitotoxicity; and axon degeneration and sprouting. We discuss how these changes relate to spontaneous oligodendrogenesis and remyelination, the evidence for and against demyelination being an important clinical problem and if remyelination contributes to motor recovery.

Cholesterol Crystals Induce Coagulation Activation through Complement-Dependent Expression of Monocytic Tissue Factor.

Cholesterol crystals (CC) are strong activators of complement and could potentially be involved in thromboinflammation through complement-coagulation cross-talk. To explore the coagulation-inducing potential of CC, we performed studies in lepirudin-based human whole blood and plasma models. In addition, immunohistological examinations of brain thrombi and vulnerable plaque material from patients with advanced carotid atherosclerosis were performed using polarization filter reflected light microscopy to identify CC. In whole blood, CC exposure induced a time- and concentration-dependent generation of prothrombin fragment 1+2 (PTF1.2), tissue factor (TF) mRNA synthesis, and monocyte TF expression. Blocking Abs against TF abolished CC-mediated coagulation, thus indicating involvement of the TF-dependent pathway. Blockade of FXII by corn trypsin inhibitor had a significant inhibitory effect on CC-induced PTF1.2 in platelet-free plasma, although the overall activation potential was low. CC exposure did not induce platelet aggregation, TF microparticle induction, or TF on granulocytes or eosinophils. Inhibition of complement C3 by CP40 (compstatin), C5 by eculizumab, or C5aR1 by PMX53 blocked CC-induced PTF1.2 by 90% and reduced TF+ monocytes from 18-20 to 1-2%. The physiologic relevance was supported by birefringent CC structures adjacent to monocytes (CD14), TF, and activated complement iC3b and C5b-9 in a human brain thrombus. Furthermore, monocyte influx and TF induction in close proximity to CC-rich regions with activated complement were found in a vulnerable plaque. In conclusion, CC could be active, releasable contributors to thrombosis by inducing monocyte TF secondary to complement C5aR1 signaling.

IL6Rα function in myeloid cells modulates the inflammatory response during irritant contact dermatitis.

Irritant contact dermatitis (ICD) is characterized by epidermal hyperplasia, infiltration of leucocytes into lesional skin and inflammatory cytokine release. The cellular infiltrate during ICD comprises primarily cells of the myeloid lineage. Our group has previously shown that the cytokine IL-6 confers a protective effect to lesional skin during ICD. How IL-6Rα function in myeloid cells is involved in the inflammatory response during ICD is, however, unknown. In the present study, utilizing a chemical model of ICD, it is shown that mice with a myeloid-specific knockout of the IL-6Rα (IL-6RαΔmyeloid ) display an exaggerated inflammatory response to benzalkonium chloride (BKC) and Jet propellant-8 (JP8) fuel, two well-characterized irritants relative to littermate control. Results from immunohistochemical and flow cytometric analyses revealed that IL-6RαΔmyeloid mouse skin displayed increased epidermal hyperplasia and inflammatory monocyte influx into lesional skin but lower numbers of resident macrophages relative to littermate controls after irritant exposure. Multiplex immunoassay revealed significantly higher levels of pro-inflammatory cytokines IL-1α and TNF-α, but reduced expression of chemokine proteins including CCL2-5, CCL7, CCL11, CXCL1 and CXCL10 in IL-6RαΔmyeloid mouse skin relative to littermate control following irritant exposure. These results highlight a previously unknown role of IL-6Rα function in myeloid cells in modulating the inflammatory response and myeloid population dynamics during ICD.

S100A9 induces nociceptive pain but is not involved in allodynia in acute experimental synovitis

Purpose: Synovitis-associated pain is an important aspect of arthritis pathology. Several inflammatory mediators released by the synovium have been implicated in the regulation of pain, including S100A8 and S100A9 which may regulate pain either via direct stimulation of TLR4 on the nerve endings in the synovium or via stimulation at the site of the dorsal root ganglia (DRG), thereby enabling an increased phagocyte infiltration, which may cause sensitization. We aimed to elucidate the role of S100A9 in the pain response after induction of an acute synovitis using streptococcal cell walls (SCW) as a trigger, comparing S100A9-/- mice and their WT controls. Methods: Synovial biopsies of 25 patients with early symptomatic OA were studied by micro-array analysis and subsequent functional annotation clustering (FAC). Patients with pain at t=5 years after biopsy isolation according to WOMAC score, were compared to those without pain. Acute synovitis was induced by a single i.a. injection of SCW in the knee joint of C57Bl6 (WT) mice and S100A9-/- mice, control mice received an i.a. saline injection. Serum S100A8/A9 levels were investigated by ELISA and expression of S100A8 and S100A9 in synovium and DRG by immunohistochemistry. Joint swelling and cell influx was assessed by 99mTc accumulaiton and histology, respectively. Pain response were investigated Incapacitance Tester (weight bearing), Catwalk (gait analysis) and von Frey’s filaments (mechanical allodynia). Gene expression of inflammatory mediators and neuron activation markers in DRG were determined by q-PCR. Monocyte influx and protein expression of NAV1.7 and GAP43 was monitored by immunohistochemistry. Results: In synovial biopsies of early symptomatic OA patients, approximately 750 genes were upregulated in patients that had pain 5 years after inclusion compared to patients without pain. Functional annotation clustering showed that inflammatory genes were highly significantly enriched (p=6,4*10-12). Among the upregulated genes were the alarmins S100A8 and S100A9. In mice, a single i.a. injection of SCW resulted in increased synovial expression of S100A8 and S100A9 and subsequent increased serum S100A8/A9 levels (2.6-fold, P < 0.001) 1 day p.i., which returned to basal levels at 7 days p.i. The increased expression of S100A9 did not contribute to the development of inflammation since joint swelling and cell influx were similar in WT and S100A9-/- mice 1 day p.i. Using the Incapacitance Tester, WT mice showed a marked and significant decrease in percentage of weight bearing on the SCW injected hindpaw (28%) compared to saline injection (47%, P < 0.001) 1 day p.i., whereas S100A9-/- mice did not. In addition, gait analysis showed that the stand-phase of the unaffected paws were significantly increased in WT mice 1 day p.i., which will reduce the load on the inflamed paw, while in S100A9-/- mice these parameters were not altered. No difference in mechanical allodynia was observed, both mouse strains showed a similar reduction of paw withdrawal threshold (4.2 and 4.5 fold decrease respectively). Analysis of DRG showed no increased phagocyte infiltration after SCW injection as determined by S100A8 and S100A9 immunohistochemistry and no change in gene expression of MCP-1, KC, IL-1β or TNF was measured. In addition, nu change in F4/80 staining was seen in both WT and S100A9-/- mice. However, expression of neuron activation markers NAV1.7, ATF3 and GAP43 were significantly increased at 1 day after SCW injection in WT mice as compared to saline injected mice (P = 0.022, 0.004 and 0.030, respectively) while SCW injection in S100A9-/- mice did not show increased expression, which is in line in with the reduced pain response observed earlier in S100A9-/- mice. Immunohistochemically the difference in NAV1.7 expression in the DRG was further confirmed at protein level. Conclusions: These findings show that S100A9, which is released from the synovium upon inflammation, is an important mediator of inflammatory nociceptive pain response in the knee, rather than by being involved in peripheral sensitization. During the acute phase of inflammation S100A8/A9 is likely regulated via direct activation of TLR4 on nerve endings in the synovium and not via increased infiltration of phagocytes in the DRG.

FPR2 regulates monocyte recruitment to promote mucosal wound repair

Mucosal wound repair is coordinated by a dynamic spatiotemporal cross talk of mediators with receptors on epithelial cells and infiltrating immune cells. After initial recruitment of neutrophils, monocytes infiltrate mucosal wounds in close proximity to repairing epithelium. Key regulators of mucosal repair include pro‐inflammatory and pro‐resolving mediators that help orchestrate temporal recruitment of immune cells, resolve acute inflammatory responses and regulate epithelial cell migration and proliferation. Immune cell derived mediators have been shown to activate G‐protein coupled receptors (GPCR) that significantly impact the inflammatory response and repair. In this report, our studies were focused on how a GPCR, formyl peptide receptor 2 (FPR2), orchestrates mucosal repair. Fpr2/3−/− mice were utilized to investigate the role of formyl‐peptide receptor 2/3 (FPR2/3), an orthologue of human FPR2/ALX, in regulating intestinal mucosal repair. Compared to wild type mice, Fpr2/3−/− mice exhibited delayed recovery from acute colitis and impaired repair after biopsy induced injury. Time course analyses of immune cell populations in the wound bed of wild type and Fpr2/3−/− mice by flow cytometry revealed mice lacking FPR2 have a reduced influx of inflammatory monocytes (defined as CD45+ CD11b+ Ly6G− Siglec F− F4/80lo Ly6Chi) on day 1 and 2 post wounding compared to wild type mice. To further supporting these data, we assessed expression of CCR2, a chemokine receptor and marker of conventional Ly6Chi inflammatory monocytes, in wounded tissue. We found reduced expression of CCR2 mRNA in tissue isolated from Fpr2/3−/− mice. Bone marrow transplant experiments revealed that monocytes from wild type mice had a competitive advantage over Fpr2/3−/− cells in migrating into healing mucosal wounds. Moreover, Fpr2/3−/− monocytes were defective in chemotaxis responses to the chemokine CCL20, a chemoattractant for monocytes that is upregulated during the early phase of inflammation. Analysis of Fpr2/3−/− monocytes revealed altered expression of the CCL20 receptor, CCR6, suggesting a role of Fpr2/3 and CCL20‐CCR6 signaling in regulating monocyte recruitment to sites of mucosal injury. In summary, these findings identify the contribution of monocyte expressed FPR2/3 in facilitating their recruitment to sites of mucosal injury to promote wound repair.Support or Funding InformationThis research was funded by DK055679, DK089763, DK059888, to AN; DK61739, DK72564, DK79392, to CAP; and a German Research Foundation Research Fellowship (SI 2282/1‐1, to DB).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Role of Jak3 in Kupffer‐cell mediated hepatic steatosis during obesity

BackgroundJanus kinase 3 (Jak3), a non‐receptor protein kinase, plays an essential role in hematopoietic cell‐growth and differentiation of intestinal epithelial mucosa. Previously, we have shown that loss of Jak3 exacerbates symptoms of obesity associated metabolic syndrome in a murine model of diet‐induced obesity through compromised intestinal barrier functions and induction of chronic low‐grade inflammation (CLGI). Though, recent studies suggest a strong linkage between CLGI and hepatic steatosis, the genetic regulation of intestinal dysbiosis and their role in hepatic steatosis is not known. In this report, we investigated the tissue‐specific role of Jak3 in maintaining hepatic homeostasis during obesity associated metabolic syndrome.MethodsIn order to test our hypothesis, we used a global Jak3 KO (Jak3−/−) mice model and compared its effect with either the intestinal epithelial specific Jak3 KO (Int Jak3 KO) mice or immune cell specific Jak3 KO (Imm Jak3 KO) mice in a model of diet‐induced obesity. The organs were collected at the end of the study period under aseptic conditions and analyzed using a combination of flowcytometric, confocal microscopy, and western analysis techniques.ResultsOur results show increased susceptibility to obesity associated metabolic syndrome in high‐fat diet treated Jak3−/− mice compared to their co‐housed littermates. Jak3−/− mice showed increased predisposition to CLGI, hepatic steatosis, increased liver to body weight ratio, and hyperglycemia compared to their co‐housed littermate controls. To determine the tissue specific role of Jak3, a high‐degree of association between CLGI and hepatic inflammation in Imm Jak3 KO mice, indicated a prominent role of hematopoietic Jak3 in maintaining Kupffer‐cell mediated hepatic steatosis. A differential flowcytometric assay showed an increased influx of monocytes, but reduced differentiation of residential Kupffer cells in imm Jak3 KO mice compared to its floxed control during diet‐induced obesity. Further, the same group of mice also showed an increased hepatic expression of TLR‐2 and TLR‐4 and a higher IL‐6, IL‐17 and Tnf‐α as compared to the control mice. Together, these results showed that a lack of Jak3 expression in immune cells leads to CLGI and a compromise in the activation of Kupffer cells coupled with increased extravasation of the circulating monocytes into the liver. This could lead to hepatic steatosis in obesity. In addition, we also found an increased Th17 effector function and simultaneous suppression of Treg cell proliferation in the absence of immune cell Jak3, when compared to its control mice, on treatment with HFD.ConclusionCollectively, these results indicate that Jak3 plays a major role in regulating the macrophage based hepatic homeostasis.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Btk inhibitor ibrutinib reduces inflammatory myeloid cell responses in the lung during murine pneumococcal pneumonia

BackgroundStreptococcus pneumoniae is a major causative agent in community-acquired pneumonia and sepsis. Overwhelming lung inflammation during pneumococcal pneumonia may hamper lung function. Ibrutinib is an irreversible inhibitor of Bruton’s tyrosine kinase (Btk), a key signaling protein controlling the activation of various immune cells, including macrophages and neutrophils. The aim of this study was to determine whether ibrutinib treatment ameliorates acute lung inflammation during pneumococcal pneumonia.MethodsMice were treated orally with ibrutinib and the effect on acute pulmonary inflammation elicited by the gram-positive bacterial cell wall component lipoteichoic acid (LTA) and during ceftriaxone-treated pneumococcal pneumonia was assessed.ResultsTreatment with ibrutinib prior to and after intranasal LTA instillation reduced alveolar macrophage activation, neutrophil influx, cytokine release and plasma leakage into the lung. Postponed treatment with ibrutinib supplementing antibiotic therapy during ongoing pneumococcal pneumonia did not impair bacterial killing in lung, blood and spleen. In this setting, ibrutinib reduced alveolar macrophage and systemic neutrophil activation and substantially diminished further monocyte and neutrophil influx in the lung. In vitro, ibrutinib inhibited macrophage TNF secretion and neutrophil activation upon LTA and pneumococcal stimulation.ConclusionsTaken together, these data indicate that the Btk inhibitor ibrutinib reduces inflammatory myeloid cell responses during acute pulmonary inflammation evoked by LTA and antibiotic-treated pneumococcal pneumonia and suggest that ibrutinib has the potential to inhibit ongoing lung inflammation in an acute infectious setting.

Monocyte-derived macrophages expand the murine stress erythropoietic niche during the recovery from anemia

AbstractAnemic stress induces a physiological response that includes the rapid production of new erythrocytes. This process is referred to as stress erythropoiesis. It is best understood in the mouse where it is extramedullary and utilizes signals and progenitor cells that are distinct from bone marrow steady-state erythropoiesis. The development of stress erythroid progenitors occurs in close association with the splenic stress erythropoiesis niche. In particular, macrophages in the niche are required for proper stress erythropoiesis. Here we show that the expansion of the niche occurs in concert with the proliferation and differentiation of stress erythroid progenitors. Using lineage tracing analysis in 2 models of anemic stress, we show that the expansion of the splenic niche is due to the recruitment of monocytes into the spleen, which develop into macrophages that form erythroblastic islands. The influx in monocytes into the spleen depends in part on Ccr2-dependent signaling mediated by Ccl2 and other ligands expressed by spleen resident red pulp macrophages. Overall, these data demonstrate the dynamic nature of the spleen niche, which rapidly expands in concert with the stress erythroid progenitors to coordinate the production of new erythrocytes in response to anemic stress.

Prenatal treatment with rosiglitazone attenuates vascular remodeling and pulmonary monocyte influx in experimental congenital diaphragmatic hernia

IntroductionExtensive vascular remodeling causing pulmonary hypertension (PH) represents a major cause of mortality in patients with congenital diaphragmatic hernia (CDH). The chemokine monocyte chemoattractant protein-1 (MCP-1) is a biomarker for the severity of PH and its activation is accompanied by pulmonary influx of monocytes and extensive vascular remodeling. MCP-1 activation can be reversed by application of rosiglitazone (thiazolidinedione). We performed this study to evaluate the role of MCP-1 for the pathogenesis of PH in experimental CDH. We hypothesized that vascular remodeling and MCP-1 activation is accompanied by pulmonary influx of fetal monocytes and can be attenuated by prenatal treatment with rosiglitazone.MethodsIn a first set of experiments pregnant rats were treated with either nitrofen or vehicle on gestational day 9 (D9). Fetal lungs were harvested on D21 and divided into CDH and control. Quantitative real-time polymerase chain reaction, Western blot (WB), and immunohistochemistry (IHC) were used to evaluate MCP-1 expression, activation, and localization. Quantification and localization of pulmonary monocytes/macrophages were carried out by IHC.In a second set of experiments nitrofen-exposed dams were randomly assigned to prenatal treatment with rosiglitazone or placebo on D18+D19. Fetal lungs were harvested on D21, divided into control, CDH+rosiglitazone, and CDH+placebo and evaluated by WB as well as IHC.ResultsIncreased thickness of pulmonary arteries of CDH fetuses was accompanied by increased systemic and perivascular MCP-1 protein expression and significantly higher amounts of pulmonary monocytes/macrophages compared to controls (p<0.01). These effects were reversed by prenatal treatment with rosiglitazone (p<0.01 vs. CDH+P; control).ConclusionPrenatal treatment with rosiglitazone has the potential to attenuate activation of pulmonary MCP-1, pulmonary monocyte influx, and vascular remodeling in experimental CDH. These results provide a basis for future research on prenatal immunomodulation as a novel treatment strategy to decrease secondary effects of PH in CDH.

The olfactory epithelium as a port of entry in neonatal neurolisteriosis

Bacterial infections of the central nervous system (CNS) remain a major cause of mortality in the neonatal population. Commonly used parenteral infection models, however, do not reflect the early course of the disease leaving this critical step of the pathogenesis largely unexplored. Here, we analyzed nasal exposure of 1-day-old newborn mice to Listeria monocytogenes (Lm). We found that nasal, but not intragastric administration, led to early CNS infection in neonate mice. In particular, upon bacterial invasion of the olfactory epithelium, Lm subsequently spread along the sensory neurons entering the brain tissue at the cribriform plate and causing a significant influx of monocytes and neutrophils. CNS infection required listeriolysin for penetration of the olfactory epithelium and ActA, a mediator of intracellular mobility, for translocation into the brain tissue. Taken together, we propose an alternative port of entry and route of infection for neonatal neurolisteriosis and present a novel infection model to mimic the clinical features of late-onset disease in human neonates.

ZIP8 induces monocyte adhesion to the aortas ex-vivo by regulating zinc influx

Monocytes recruited and adhering to the inflamed arteries are crucial for atherosclerosis development. Here, we report the role of zinc (Zn2+) homeostasis in monocyte adhesion and recruitment. By comparing the expression levels of Zn2+ transporters between non-adhering and adhering monocytes, we found that the Zn2+ importer ZIP8 was specifically upregulated in monocytes adhering to the aortas ex-vivo. Although the overexpression of ZIP8 increased the absorption of Zn2+, Fe2+ and Cd2+ in monocytes, only Zn2+ supplementation was demonstrated capable of promoting the adhesion of monocytes to endothelial monolayers in vitro. In addition, we confirmed the role of ZIP8-dependent Zn2+ influx in promoting monocyte adhesion to the aortas ex-vivo. More importantly, the enforced expression of ZIP8 increased monocyte adhesion and recruitment to the nascent atherosclerotic lesions in ApoE−/− mice. Overall, our results suggest that the Zn2+ influx in monocytes regulated by ZIP8 is a novel factor determining their adhesion and recruitment to atherosclerotic lesions, and that targeting ZIP8 or Zn2+ homeostasis may represent a novel strategy to interfere these activities.

Abstract 67: Chemotherapy induced pro-metastatic changes in the primary breast tumors of racially diverse patients

Abstract Neoadjuvant chemotherapy (NAC) induces influx of bone marrow-derived proangiogenic Tie2hi monocytes into the primary tumor, resulting in increased density of perivascular Tie2hi macrophages (1). Perivascular Tie2hi/Vegfhi macrophages in physical contact with Mena expressing cancer cells create micro-anatomic sites of transient vascular permeability called TMEM, which mediate cancer cell intravasation and dissemination (2). Cancer cells capable of intravasation via TMEM sites express high level of MenaINV, an isoform of Mena, induced by macrophage contact, which renders tumor cells intravasation-competent (3, 4). Consequently, breast cancers from mice and patients treated with NAC have increased density of TMEM sites and show increased expression of MenaINV (1) Moreover, in PyMT mouse mammary carcinoma and patient derived xenografts, NAC increases the number of circulating tumor cells and lung metastasis (1). The Tie2-inhibitor rebastinib, which inhibits TMEM function, can reverse chemotherapy-induced increases in the number of CTCs and lung metastasis in mouse mammary carcinoma (1, 5). Although NAC induces pro-metastatic changes in breast cancer microenvironment, large randomized prospective studies did not find significant differences in distant-recurrence free survival (DRFS) and overall survival (OS) between breast cancer patients treated with adjuvant and neoadjuvant chemotherapy in predominantly white populations. Since the breast cancer microenvironment in black women has higher microvascular density and density of Tie2hi macrophages, suggesting that black women may be more prone to develop TMEM-associated pro-metastatic changes in response to NAC, we questioned if there is a difference in DRFS in black women treated with NAC compared to AC. We evaluated DRFS in 1,211 racially diverse patients with localized or regionally advanced breast cancer treated with neoadjuvant or adjuvant chemotherapy between January 2000 and December 2016 and found that black patients with localized breast cancer treated with systemic neoadjuvant chemotherapy not only have inferior DRFS compared to white patients, but also worse DRFS when compared to black patients treated with adjuvant chemotherapy, after adjustment for clinical covariates in multivariate analysis. The biologic factors contributing to this finding, in particular TMEM-mediated pro-metastatic changes have been evaluated and will be discussed. 1. Karagiannis et al, Sci Transl Med. 2017;9:397. 2. Harney et al, Cancer discovery. 2015;5:932. 3. Pignatelli J et al, Sci Rep. 2016;6:37874. 4. Pignatelli J et al, Sci Signal. 2014;7:353. 5. Harney et al, Mol Cancer Ther. 2017;16:2486. Citation Format: George S. Karagiannis, Jessica M. Pastoriza, Sonali Lanjawar, Yarong Wang, David Entenberg, Esther Cheng, Timothy M. Dalfonso, Joan G. Jones, Jesus Anampa, Thomas E. Rohan, Joseph A. Sparano, John S. Condeelis, Maja H. Oktay. Chemotherapy induced pro-metastatic changes in the primary breast tumors of racially diverse patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 67.