Resident Mφ Research Articles

Inflammation‐Activated Endogenous Macrophage‐Mediated Prodrug Delivery System Overcoming Biological Barriers for Enhanced Oral Meningitis Therapy

AbstractSignificant health risks are posed by meningitis due to its rapid progression, and challenges are encountered in intravenous antibiotic administration, especially in crossing the blood‐brain barrier. To address this, an inflammation‐activated, endogenous macrophage (MΦ)‐mediated oral prodrug delivery system is developed for targeted therapeutic interventions in bacterial meningitis treatment. This system is guided by inflammation‐derived chemoattractants and triggers drug release through inflammation‐induced reactive oxygen species (ROS). Comprised of naturally derived β‐glucans conjugated with the antibiotic cefotaxime (CTX) using a ROS‐responsive linker, nanoparticles (βGlus–CTX NPs) are formed in aqueous solutions. In a mouse model of Klebsiella pneumoniae‐induced meningitis, orally administered βGlus–CTX NPs are traversed by intestinal microfold cells, surpassing the intestine‐epithelial barrier, and are absorbed by resident endogenous MΦ. These MΦ‐mediated drug delivery vehicles are then traveled through the lymphatic and circulatory systems, crossing the compromised blood‐brain barrier, ultimately reaching inflamed brain tissues, guided by their derived chemoattractants. In ROS‐rich inflamed tissue environments, the linkers in the βGlus–CTX NPs are cleaved, releasing therapeutic CTX for localized treatment. Targeted antibiotic treatment for bacterial meningitis is offered by this oral, endogenous MΦ‐mediated prodrug delivery system, overcoming the robust gut‐to‐brain biological barriers and potentially enhancing effectiveness for comfortable home‐based treatment.

Microglia at the blood brain barrier in health and disease.

The blood brain barrier (BBB) plays a crucial role in maintaining brain homeostasis by selectively preventing the entry of substances from the peripheral blood into the central nervous system (CNS). Comprised of endothelial cells, pericytes, and astrocytes, this highly regulated barrier encompasses the majority of the brain's vasculature. In addition to its protective function, the BBB also engages in significant crosstalk with perivascular macrophages (MΦ) and microglia, the resident MΦ of the brain. These interactions play a pivotal role in modulating the activation state of cells comprising the BBB, as well as MΦs and microglia, themselves. Alterations in systemic metabolic and inflammatory states can promote endothelial cell dysfunction, reducing the integrity of the BBB and potentially allowing peripheral blood factors to leak into the CNS compartment. This may mediate activation of perivascular MΦs, microglia, and astrocytes, and initiate further immune responses within the brain parenchyma, suggesting neuroinflammation can be triggered by signaling from the periphery, without primary injury or disease originating within the CNS. The intricate interplay between the periphery and the CNS through the BBB highlights the importance of understanding the role of microglia in mediating responses to systemic challenges. Despite recent advancements, our understanding of the interactions between microglia and the BBB is still in its early stages, leaving a significant gap in knowledge. However, emerging research is shedding light on the involvement of microglia at the BBB in various conditions, including systemic infections, diabetes, and ischemic stroke. This review aims to provide a comprehensive overview of the current research investigating the intricate relationship between microglia and the BBB in health and disease. By exploring these connections, we hope to advance our understanding of the role of brain immune responses to systemic challenges and their impact on CNS health and pathology. Uncovering these interactions may hold promise for the development of novel therapeutic strategies for neurological conditions that involve immune and vascular mechanisms.

CD83 acts as immediate early response gene in activated macrophages and exhibits specific intracellular trafficking properties

Macrophages (MΦ) are remarkably plastic cells, which assume phenotypes in every shade between a pro-inflammatory classical activation, and anti-inflammatory or resolving activation. Therefore, elucidation of mechanisms involved in shaping MΦ plasticity and function is key to understand their role during immunological balance. The immune-modulating CD83 molecule is expressed on activated immune cells and various tissue resident MΦ, rendering it an interesting candidate for affecting MΦ biology. However, in-depth analyses of the precise kinetics and trafficking of CD83 within pro-inflammatory, LPS activated bone-marrow-derived MΦ have not been performed. In this study, we show that activation with LPS leads to a very fast and strong, but transient increase of CD83 expression on these cells. Its expression peaks within 2 h of stimulation and is thereby faster than the early activation antigen CD69. To trace the CD83 trafficking through MΦs, we employed multiple inhibitors, thereby revealing a de novo synthesis and transport of the protein to the cell surface followed by lysosomal degradation, all within 6 h. Moreover, we found a similar expression kinetic and trafficking in human monocyte derived MΦ. This places CD83 at a very early point of MΦ activation suggesting an important role in decisions regarding the subsequent cellular fate.

Origin of M2 Mϕ and its macrophage polarization by TGF-β in a mice intervertebral injury model.

IntroductionStudies have identified the presence of M1 and M2 macrophages (Mϕ) in injured intervertebral discs (IVDs). However, the origin and polarization-regulatory factor of M2 Mϕ are not fully understood. TGF-β is a regulatory factor for M2 polarization in several tissues. Here, we investigated the source of M2 Mϕ and the role of TGF-β on M2 polarization using a mice disc-puncture injury model.MethodsTo investigate the origin of M2 macrophages, 30 GFP chimeric mice were created by bone marrow transplantation. IVDs were obtained from both groups on pre-puncture (control) and post-puncture days 1, 3, 7, and 14 and CD86 (M1 marker)- and CD206 (M2 marker)-positive cells evaluated by flow cytometry (n = 5 at each time point). To investigate the role of TGF-β on M2 polarization, TGF-β inhibitor (SB431542) was also injected on post-puncture days (PPD) 5 and 6 and CD206 expression was evaluated on day 7 by flow cytometry (n = 5) and real time PCR (n = 10).ResultsThe proportion of CD86+ Mϕ within the GFP+ population was significantly increased at PPD 1, 3, 7, and 14 compared to control. CD206-positive cells in GFP-populations were significantly increased on PPD 7 and 14. In addition, the percentage of CD206-positive cells was significantly higher in GFP-populations than in GFP+ populations. TGF-β inhibitor reduced CD206-positive cells and Cd206 expression at 7 days after puncture.ConclusionOur findings suggest that M2 Mϕ following IVD injury may originate from resident Mϕ. TGF-β is a key factor for M2 polarization of macrophages following IVD injury.

Novel insights into the electrophysiology of murine cardiac macrophages: relevance of voltage-gated potassium channels.

AimsMacrophages (MΦ), known for immunological roles, such as phagocytosis and antigen presentation, have been found to electrotonically couple to cardiomyocytes (CM) of the atrioventricular node via Cx43, affecting cardiac conduction in isolated mouse hearts. Here, we characterize passive and active electrophysiological properties of murine cardiac resident MΦ, and model their potential electrophysiological relevance for CM.Methods and resultsWe combined classic electrophysiological approaches with 3D florescence imaging, RNA-sequencing, pharmacological interventions, and computer simulations. We used Cx3 mice wherein cardiac MΦ are fluorescently labelled. FACS-purified fluorescent MΦ from mouse hearts were studied by whole-cell patch-clamp. MΦ electrophysiological properties include: membrane resistance 2.2±0.1 GΩ (all data mean±SEM), capacitance 18.3±0.1 pF, resting membrane potential −39.6±0.3 mV, and several voltage-activated, outward or inwardly rectifying potassium currents. Using ion channel blockers (barium, TEA, 4-AP, margatoxin, XEN-D0103, and DIDS), flow cytometry, immuno-staining, and RNA-sequencing, we identified Kv1.3, Kv1.5, and Kir2.1 as channels contributing to observed ion currents. MΦ displayed four patterns for outward and two for inward-rectifier potassium currents. Additionally, MΦ showed surface expression of Cx43, a prerequisite for homo- and/or heterotypic electrotonic coupling. Experimental results fed into development of an original computational model to describe cardiac MΦ electrophysiology. Computer simulations to quantitatively assess plausible effects of MΦ on electrotonically coupled CM showed that MΦ can depolarize resting CM, shorten early and prolong late action potential duration, with effects depending on coupling strength and individual MΦ electrophysiological properties, in particular resting membrane potential and presence/absence of Kir2.1.ConclusionOur results provide a first electrophysiological characterization of cardiac resident MΦ, and a computational model to quantitatively explore their relevance in the heterocellular heart. Future work will be focussed at distinguishing electrophysiological effects of MΦ–CM coupling on both cell types during steady-state and in patho-physiological remodelling, when immune cells change their phenotype, proliferate, and/or invade from external sources.

Resident macrophages acquire innate immune memory in staphylococcal skin infection.

Staphylococcus aureus (S. aureus) is a common colonizer of healthy skin and mucous membranes. At the same time, S. aureus is the most frequent cause of skin and soft tissue infections. Dermal macrophages (Mφ) are critical for the coordinated defense against invading S. aureus, yet they have a limited life span with replacement by bone marrow derived monocytes. It is currently poorly understood whether localized S. aureus skin infections persistently alter the resident Mφ subset composition and resistance to a subsequent infection. In a strictly dermal infection model we found that mice, which were previously infected with S. aureus, showed faster monocyte recruitment, increased bacterial killing and improved healing upon a secondary infection. However, skin infection decreased Mφ half-life, thereby limiting the duration of memory. In summary, resident dermal Mφ are programmed locally, independently of bone marrow-derived monocytes during staphylococcal skin infection leading to transiently increased resistance against a second infection.

Recruitment of hepatic macrophages from monocytes is independent of IL-4Rα but is associated with ablation of resident macrophages in schistosomiasis.

Alternatively activated Mφs (AAMφ) accumulate in hepatic granulomas during schistosomiasis and have been suggested to originate in the bone marrow. What is less understood is how these Mφ responses are regulated after S. mansoni infection. Here, we investigated the role of IL-4 receptor α-chain (IL-4Rα)-signalling in the dynamics of liver Mφ responses. We observed that IL-4Rα signalling was dispensable for the recruitment of Ly6Chi monocytes and for their conversion into F4/80hi CD64hi CD11bhi Mφ. Moreover, while IL-4Rα provided an AAMφ phenotype to liver F4/80hi CD64hi CD11bhi Mφ that was associated with regulation of granuloma formation, it was dispensable for host survival. Resident F4/80hi CD64hi CD11blo Mφ did not upregulate the AAMφ signature gene Ym1. Rather, resident Mφ nearly disappeared by week 8 after infection and artificial ablation of resident Mφ in CD169DTR mice did not affect the response to S. mansoni infection. Interestingly, ablation of CD169+ cells in naive mice resulted in the accumulation of F4/80hi CD64hi CD11bhi Mφ, which was amplified when ablation occurred during schistosomiasis. Altogether, our results suggest the ablation of resident KCs after S. mansoni infection to be associated with the recruitment and accumulation of F4/80hi CD64hi CD11bhi Mφ with lyz2-dependent IL-4Rα contributing to the regulation of granuloma inflammation but being dispensable for host survival.

Changes in duodenal CD163-positive cells in dogs with chronic enteropathy after successful treatment

Chronic enteropathy (CE) in dogs is characterized retrospectively per treatment response as food-responsive enteropathy (FRE), antibiotic-responsive enteropathy (ARE), and immunosuppressant-responsive enteropathy (IRE) – the latter most resembling inflammatory bowel disease in people. The aim of this study was to characterize duodenal macrophages (Mϕ) in CE using immunohistochemistry; with calprotectin (CAL) as a marker of early differentiated Mϕ and CD163 expression as a marker for resident Mϕ in the duodenum before and after treatment. Prior to treatment, dogs with FRE and IRE had a lower CD163+/CAL+ ratio than control dogs (CTRL) in crypts; this increased significantly and normalized compared with CTRL after treatment. Conversely, the CD163+/CAL+ ratio in dogs with ARE was comparable to that in healthy dogs before and after treatment. In summary, these results suggest that Mϕ play a role in the pathogenesis of CE in FRE and IRE, with a decrease in resident Mϕ and an increase in early differentiated Mϕ, but not in ARE dogs. Mϕ normalize after successful treatment.

Functional macrophages and gastrointestinal disorders.

Macrophages (MΦ) differentiate from blood monocytes and participate in innate and adaptive immunity. Because of their abilities to recognize pathogens and activate bactericidal activities, MΦ are always discovered at the site of immune defense. MΦ in the intestine are unique, such that in the healthy intestine, they possess complex mechanisms to protect the gut from inflammation. In these complex mechanisms, they produce anti-inflammatory cytokines, such as interleukin-10 and transforming growth factor-β, and inhibit the inflammatory pathways mediated by Toll-like receptors. It has been demonstrated that resident MΦ play a crucial role in maintaining intestinal homeostasis, and they can be recognized by their unique markers. Nonetheless, in the inflamed intestine, the function of MΦ will change because of environmental variation, which may be one of the mechanisms of inflammatory bowel disease (IBD). We provide further explanation about these mechanisms in our review. In addition, we review recent discoveries that MΦ may be involved in the development of gastrointestinal tumors. We will highlight the possible therapeutic targets for the management of IBD and gastrointestinal tumors, and we also discuss why more details are needed to fully understand all other effects of intestinal MΦ.

Immunohistochemical characterization of gastrointestinal macrophages/phagocytes in dogs with inflammatory bowel disease (IBD) and non-IBD dogs

Intestinal Mϕ play a pivotal role in the maintenance of gut homeostasis, but can also contribute to inflammation such as inflammatory bowel disease (IBD). In contrast to human tissues, little is known about phenotypes of Mϕ in the canine gastrointestinal tract. Therefore, an immunohistochemical study was performed using Abs against Mϕ-associated molecules (Cluster of differentiation (CD)64, CD163, CD204, ionized calcium-binding adaptor molecule 1, L1 Ag, and MHC II) on stomach, duodenum, jejunum, ileum and colon from non-IBD dogs. In addition, marker-expression in the stomach, duodenum and colon of the non-IBD dogs was compared to that in dogs with IBD. Results revealed predominance of resident Mϕ displaying an anti-inflammatory phenotype represented by expression of CD163 as well as CD204 in the gut of non-IBD dogs with high Mϕ numbers especially present in the small intestinal villus area. Compared to non-IBD tissue counterparts, stomach, duodenum, and colon from dogs with IBD showed reduced Mϕ numbers with the exception of slightly increased numbers of CD64+ Mϕ. Correlation analyses between marker-expression of Mϕ and the Canine Inflammatory Bowel Disease Activity Index as well as histological scores failed to reveal relevant relationships. The present study provides evidence of the canine steady state gastrointestinal tract being dominated by Mϕ with anti-inflammatory properties maintaining gut homeostasis. A significant reduction in these resident Mϕ may reflect disturbances in homeostatic capacity that could contribute to the development of canine IBD. In contrast to human IBD and murine disease models, infiltration of pro-inflammatory Mϕ does not significantly contribute to the inflammatory process of canine IBD, which may illustrate possible species-specific differences in IBD pathogenesis.

The Biology of Monocytes and Dendritic Cells: Contribution to HIV Pathogenesis.

Myeloid cells such as monocytes, dendritic cells (DC) and macrophages (MΦ) are key components of the innate immune system contributing to the maintenance of tissue homeostasis and the development/resolution of immune responses to pathogens. Monocytes and DC, circulating in the blood or infiltrating various lymphoid and non-lymphoid tissues, are derived from distinct bone marrow precursors and are typically short lived. Conversely, recent studies revealed that subsets of tissue resident MΦ are long-lived as they originate from embryonic/fetal precursors that have the ability to self-renew during the life of an individual. Pathogens such as the human immunodeficiency virus type 1 (HIV-1) highjack the functions of myeloid cells for viral replication (e.g., MΦ) or distal dissemination and cell-to-cell transmission (e.g., DC). Although the long-term persistence of HIV reservoirs in CD4+ T-cells during viral suppressive antiretroviral therapy (ART) is well documented, the ability of myeloid cells to harbor replication competent viral reservoirs is still a matter of debate. This review summarizes the current knowledge on the biology of monocytes and DC during homeostasis and in the context of HIV-1 infection and highlights the importance of future studies on long-lived resident MΦ to HIV persistence in ART-treated patients.

Myeloid cell recruitment versus local proliferation differentiates susceptibility from resistance to filarial infection.

Both TH2-dependent helminth killing and suppression of the TH2 effector response have been attributed to macrophages (MΦ) activated by IL-4 (M(IL-4)). To investigate how M(IL-4) contribute to diverse infection outcomes, the MΦ compartment of susceptible BALB/c mice and more resistant C57BL/6 mice was profiled during infection of the pleural cavity with the filarial nematode, Litomosoides sigmodontis. C57BL/6 mice exhibited a profoundly expanded resident MΦ (resMΦ) population, which was gradually replenished from the bone marrow in an age-dependent manner. Infection status did not alter the bone-marrow derived contribution to the resMΦ population, confirming local proliferation as the driver of resMΦ expansion. Significantly less resMΦ expansion was observed in the susceptible BALB/c strain, which instead exhibited an influx of monocytes that assumed an immunosuppressive PD-L2+ phenotype. Inhibition of monocyte recruitment enhanced nematode killing. Thus, the balance of monocytic vs. resident M(IL-4) numbers varies between inbred mouse strains and impacts infection outcome.

Deletion of c-FLIP from CD11bhi Macrophages Prevents Development of Bleomycin-induced Lung Fibrosis.

Idiopathic pulmonary fibrosis is a progressive lung disease with complex pathophysiology and fatal prognosis. Macrophages (MΦ) contribute to the development of lung fibrosis; however, the underlying mechanisms and specific MΦ subsets involved remain unclear. During lung injury, two subsets of lung MΦ coexist: Siglec-Fhi resident alveolar MΦ and a mixed population of CD11bhi MΦ that primarily mature from immigrating monocytes. Using a novel inducible transgenic system driven by a fragment of the human CD68 promoter, we targeted deletion of the antiapoptotic protein cellular FADD-like IL-1β-converting enzyme-inhibitory protein (c-FLIP) to CD11bhi MΦ. Upon loss of c-FLIP, CD11bhi MΦ became susceptible to cell death. Using this system, we were able to show that eliminating CD11bhi MΦ present 7-14 days after bleomycin injury was sufficient to protect mice from fibrosis. RNA-seq analysis of lung MΦ present during this time showed that CD11bhi MΦ, but not Siglec-Fhi MΦ, expressed high levels of profibrotic chemokines and growth factors. Human MΦ from patients with idiopathic pulmonary fibrosis expressed many of the same profibrotic chemokines identified in murine CD11bhi MΦ. Elimination of monocyte-derived MΦ may help in the treatment of fibrosis. We identify c-FLIP and the associated extrinsic cell death program as a potential pathway through which these profibrotic MΦ may be pharmacologically targeted.

Abstract 21026: Macrophages Preferentially Endocytose Systemically-Delivered Exosomes at Steady State or During Acute Inflammation

Introduction: Cardiosphere-derived cells (CDCs), which are in advanced clinical testing for heart failure, Duchenne muscular dystrophy and pulmonary hypertension, secrete exosomes (CDC exo ) which are highly anti-inflammatory. CDC exo , but not fibroblast (FB) exosomes, lead to cardioprotection through polarization of macrophages (Mφ) following ischemia/reperfusion injury (de Couto et al., Circulation, 2017). Here we demonstrate that intravenous delivery of CDC exo in mice (healthy or acutely inflamed) leads to preferential uptake by resident Mφ via phagocytosis. Methods & Results: To assess biodistribution, we labeled CDC exo with the fluorescent liposomal dye DiD (Invitrogen). Healthy mice (C57Bl6; 8-12 weeks) were treated with an intraperitoneal injection of either LPS (5mg/kg) or saline prior to intravenous infusion of DiD-labeled CDC exo (in 100μL saline). Four hours later, organs were collected for ex vivo analysis. Biofluorescence imaging (Xenogen) revealed strong DiD signal in Mφ-rich organs including the liver, spleen, and lung, while immunohistochemistry and flow cytometry resolved preferential uptake of CDC exo by tissue Mφ within the liver (60-80% DiD + CD11b + ), spleen (70-80% DiD + F4/80 + ), lung (60-90% DiD + F4/80 + ), and peritoneal cavity (40-60% DiD + CD11b + ). The heart also exhibited measurable fluorescence signals. To determine how avidly distinct cardiovascular cell types preferentially take up CDC exo in vitro , Mφ, FB, endothelial cells (EC), and cardiomyocytes (CM) were exposed to DiD-labeled CDC exo and examined over time. Within one hour, Mφ demonstrated the greatest uptake of CDC exo (Mφ: 81%; FB: 60%; EC: 65%; CM: 51%; p<0.05). The addition of both phagocytosis (Cytochalasin D) and clathrin (Pitstop 2) inhibitors completely blocked CDC exo uptake by Mφ. Conclusions: Intravenous delivery of CDC exo in mice (healthy or acutely inflamed) leads to preferential uptake by Mφ within organs that are rich in such cells (i.e., liver, spleen, lung, and peritoneal cavity). In vitro , Mφ take up CDC exo via phagocytosis and endocytosis more readily than other cardiac cell types. These data suggest the potential utility of CDC exo treatment in cardiovascular diseases where Mφ infiltration is prominent (e.g., atherosclerosis).

A Novel Subset of Anti-Inflammatory CD138+ Macrophages Is Deficient in Mice with Experimental Lupus.

Dead cells accumulating in the tissues may contribute to chronic inflammation. We examined the cause of impaired apoptotic cell clearance in human and murine lupus. Dead cells accumulated in bone marrow from lupus patients but not from nonautoimmune patients undergoing myeloablation, where they were efficiently removed by macrophages (MΦ). Impaired apoptotic cell uptake by MΦ also was seen in mice treated i.p. with pristane (develop lupus) but not mineral oil (MO) (do not develop lupus). The inflammatory response to both pristane and MO rapidly depleted resident (Tim4+) large peritoneal MΦ. The peritoneal exudate of pristane-treated mice contained mainly Ly6Chi inflammatory monocytes; whereas in MO-treated mice, it consisted predominantly of a novel subset of highly phagocytic MΦ resembling small peritoneal MΦ (SPM) that expressed CD138+ and the scavenger receptor Marco. Treatment with anti-Marco-neutralizing Abs and the class A scavenger receptor antagonist polyinosinic acid inhibited phagocytosis of apoptotic cells by CD138+ MΦ. CD138+ MΦ expressed IL-10R, CD206, and CCR2 but little TNF-α or CX3CR1. They also expressed high levels of activated CREB, a transcription factor implicated in generating alternatively activated MΦ. Similar cells were identified in the spleen and lung of MO-treated mice and also were induced by LPS. We conclude that highly phagocytic, CD138+ SPM-like cells with an anti-inflammatory phenotype may promote the resolution of inflammation in lupus and infectious diseases. These SPM-like cells are not restricted to the peritoneum and may help clear apoptotic cells from tissues such as the lung, helping to prevent chronic inflammation.

Effect of optimal linear control in human monocytes and macrophages system

Monocytes M and macrophages Mϕ cells are mononuclear essential components of the innate immune system response. M and Mϕ are mononuclear phagocytes that have crucial roles in tissue homeostasis and innate immunity. M Are key players during inflammation and pathogen challenge also are precursor of Mϕ, whereas tissue resident Mϕ has important functions in development, tissue homeostasis and the resolution of inflammation. In previous work, linear optimal control equations with tracking in x2(t) (this is Mϕ) and control only in x1(t), (this is M) were applied obtaining good results. In this work, Linear Optimal Control equations for states x1(t) and x2(t), with tracking in the state x2(t) were applied. It is possible to obtain better convergence results of the states to the asymptotic values (healthy state) when the production rate of (M) and λ into the state equations was modified. Tables and graphics are included.

Apoptotic cell driven ROS burst drives AhR dependent immunologic tolerance and suppression of lupus

Abstract Tissue-resident macrophages (MΦ) are crucial in driving tolerance and preventing systemic autoimmunity. We have previously shown that exposure to apoptotic cells triggers a regulatory circuit dependent on IL-10 production in resident MΦ. However, key molecular mechanisms driving the regulatory response to apoptosis are not clear. RNA transcriptome analysis of MΦs after exposure to apoptotic cells identified strong transcript association with the aryl hydrocarbon receptor (AhR) signaling pathway, an association that was confirmed by phenotypic and biochemical analysis. When AhR activity was blocked, apoptotic cells induced an alteration in the mRNA signature enhancing proinflammatory effector expression. Functional analysis revealed that the DNA from apoptotic cells activated AhR in a reactive oxygen species (ROS) dependent mechanism and AhR is required for IL-10 production. Consequently, inhibition or deletion of AhR signals fundamentally altered immune responses to apoptotic cells in vivo resulting in proinflammatory cytokine production, increased effector T cell responses, and failure of long-term tolerance to apoptotic cell-associated antigens. Surprisingly, mice lacking AhR developed progressive systemic autoimmunity characterized by excessive MΦ and lymphocyte activation and renal pathology. Similarly, SLE-prone mice treated with AhR antagonist exhibited poor survival, while agonist treatment ablated disease pathology. Finally, an AhR transcriptional signature was significantly associated with active SLE flare in SLE patients. Thus, the data demonstrates the AhR pathway is a key molecular circuit responsible for apoptotic cell driven tolerance and suppression of inflammatory autoimmunity.

CD4+ T cell control of Salmonella infection and persistence is dependent on different macrophage subsets

Abstract The control of the persistent phagosomal pathogen, Salmonella enterica (Se), by the adaptive immune system is dependent on pathogen-specific CD4+ T-helper 1 (Th1) cells. To explore the hypothesis that Se uses a specific cellular niche to evade Th1 cells in order to persist, we generated a strain of Se that expresses a chromosomally-encoded dTomato protein that displays bright and stable fluorescence in vivo. Following oral infection in 129sv/J mice we found dTomato+ cells in macrophage (MΦ), monocyte, and neutrophil populations. Depletion of neutrophils during infection had no effect on bacterial burden or survival. In contrast, depletion of monocytes and MΦ in CCR2 or LysM-Cre CD115 stop-flox diphtheria toxin (DT) receptor transgenic mice increased bacterial counts and death 6–8 days after DT treatment, indicating an important role for these cells in maintaining Se persistence. Depletion of CD4+ T cells led to increases in bacterial counts and an altered infected cell profile compared to control animals. The frequency of infected CD11bHi MerTKInt infiltrating MΦ was increased, while infected CD11bIntMerTKHi tissue resident MΦ remained relatively constant, suggesting that Th1 cells interact preferentially with infiltrating MΦ. Infected tissue resident MΦ expressed less MHCII, CD86 and more PD-L1 and PD-L2 than uninfected counterparts, and a reciprocal expression pattern was observed on infected infiltrating MΦ. These data suggest that Th1 cells promote killing of intracellular Se in infiltrating MΦ and either ignore or are inhibited by infected tissue resident MΦ providing a reservoir where Se can persist for the lifetime of the infected animal.

Implications for the role of macrophages in a model of myocardial fibrosis: CCR2(-/-) mice exhibit an M2 phenotypic shift in resident cardiac macrophages.

Macrophages (MΦ) are functionally diverse and dynamic. Until recently, cardiac MΦ were assumed to be monocyte derived; however, resident cardiac MΦ (rCMΦ), present at baseline, were identified in myocardia and have been implicated in cardiac healing. Previously, we demonstrated that CCR2(-/-) mice are protected from myocardial fibrosis - an observation initially attributed to changes in infiltrating monocytes. Here, we reexplored this observation in the context of our new understanding of rCMΦ. Male CCR2(-/-) and C57BL/6 hearts were digested and purified to a single cell suspension, incubated with fluorophore-linked antibodies (CCR2, CX3CR1, CD11b, Ly6C, TNF-α, and IL-10), and assessed by flow cytometry. Differentiated MΦ were cocultured with fibroblasts in order to characterize how MΦ phenotype influences fibroblast activation. Fibroblasts were characterized for their expression of smooth muscle cell actin (SMA). A significant decrease in Ly6C expression was observed in the CCR2(-/-) cardiac MΦ population relative to WT, which corresponded with significantly lower TNF-α expression and significantly higher IL-10 expression. Using in vitro coculture system, classical MΦ promoted fibroblast activation relative to nonclassical MΦ. CCR2(-/-) rCMΦ favor a more antiinflammatory phenotype relative to WT controls. Moreover, a shift toward the antiinflammatory promotes proliferation, but not activation in vitro. Together, these observations suggest that antiinflammatory cardiac MΦ populations may inhibit myocardial fibrosis in a pathological setting by preventing the activation of fibroblasts. Here, we provide novel evidence for baseline differences in rCMΦ phenotypes (i.e. classical vs. nonclassical) and how these differences could modulate cardiac healing. Importantly, we observed differences in how classical vs. nonclassical MΦ influenced fibroblast activation, which could, in turn, affect fibrosis.

The anti‐inflammatory effects of Spirulina platensis extract are mediated, in part, through the induction of an endotoxin tolerance‐like mechanism

Endotoxin tolerance is a phenomenon where exposure of innate immune cells to lipopolysaccharide (LPS) induces a refractory state to subsequent endotoxin exposures, which is characterized by a lack of pro‐inflammatory cytokine and chemokine production. We previously showed that pretreatment of macrophages (MΦ) with Spirulina platensis organic extract (SPE) repressed LPS‐induced inflammatory gene expression and cytokine secretion through the inhibition of nuclear factor κB p65 nuclear translocation. We also demonstrated that splenocytes and resident peritoneal MΦ isolated from C57BL/6J mice fed a high fat/high sucrose (HF/HS) diet containing 0.25% (w/w) SPE for 16 weeks displayed significantly attenuated expression of pro‐inflammatory genes when they were stimulated ex vivo by LPS compared to those from mice on a HF/HS control diet. The objective of this study was to investigate if SPE ameliorates LPS‐induced inflammatory gene expression in MΦ via an endotoxin tolerance‐like mechanism. Compared to naïve MΦ exposed to LPS for the first time, endotoxin tolerant (ET) RAW 264.7 MΦ, bone marrow‐derived MΦ, and THP‐1 MΦ showed significantly less interleukin (IL)‐1β expression after stimulation with 100 ng/mL of LPS, which was also observed with SPE pretreatment. The expression of IL‐6, tumor necrosis factor α, and monocyte chemoattractant protein‐1 was also repressed in ET as well as SPE‐pretreated RAW 264.7 MΦ. Consistent with the repressed expression of pro‐inflammatory genes, the translocation of p65 into the nucleus was markedly reduced in both ET and SPE‐pretreated MΦ upon LPS stimulation. It has been demonstrated that nuclear p50 homodimer and B‐cell lymphoma 3‐encoded protein (BCL3) are increased in monocytes and MΦ with endotoxin tolerance, consequently attenuating p65 transcriptional activity. We found that both SPE‐pretreated and ET RAW 264.7 MΦ had increased nuclear protein levels of p50 and BCL3. Quantification of nuclear p50 and p65 proteins showed that in ET and SPE‐pretreated cells, p50/p65 ratios were increased by more than 3‐fold compared to naïve MΦ when they were stimulated with LPS. In addition, ET MΦ showed significantly increased expression of BCL3, p105, inhibitor of κBα and IL‐ receptor‐associated kinase M, all of which are shown to be increased in endotoxin tolerance. In contrast to the LPS‐induced endotoxin tolerance, SPE significantly increased only BCL3 and p105 expression. At the protein level, endotoxin tolerization increased nuclear p50 proteins, but not BCL3, while SPE increased the nuclear abundance of both. In conclusion, the anti‐inflammatory effect of SPE is at least partly attributable to the induction of an endotoxin tolerance‐like state in macrophages, which shares common characteristics of MΦ endotoxin tolerance, while features distinctive from endotoxin tolerance also exist with SPE treatment.Support or Funding InformationUSDA AFRI (2014‐01870)