Toxoplasma gondii Profilin Promotes Recruitment of Ly6Chi CCR2+ Inflammatory Monocytes That Can Confer Resistance to Bacterial Infection

Ly6C⁺ inflammatory monocytes are essential to host defense against Toxoplasma gondii, Listeria monocytogenes and other infections. During T. gondii infection impaired inflammatory monocyte emigration results in severe inflammation and failure to control parasite replication. However, the T. gondii factors that elicit these monocytes are unknown. Early studies from the Remington laboratory showed that mice with a chronic T. gondii infection survive lethal co-infections with unrelated pathogens, including L. monocytogenes, but a mechanistic analysis was not performed. Here we report that this enhanced survival against L. monocytogenes is due to early reduction of bacterial burdens and elicitation of Ly6C⁺ inflammatory monocytes. We demonstrate that a single TLR11/TLR12 ligand profilin (TgPRF) was sufficient to reduce bacterial burdens similar to T. gondii chronic infection. Stimulation with TgPRF was also sufficient to enhance animal survival when administered either pre- or post-Listeria infection. The ability of TgPRF to reduce L. monocytogenes burdens was dependent on TLR11 and required IFN-γ but was not dependent on IL-12 signaling. TgPRF induced rapid production of MCP-1 and resulted in trafficking of Ly6C^hi CCR2⁺ inflammatory monocytes and Ly6G⁺ neutrophils into the blood and spleen. Stimulation with TgPRF reduced L. monocytogenes burdens in mice depleted with the Ly6G specific MAb 1A8, but not in Ly6C/Ly6G specific RB6-8C5 depleted or CCR2^−/− mice, indicating that only inflammatory monocytes are required for TgPRF-induced reduction in bacterial burdens. These results demonstrate that stimulation of TLR11 by TgPRF is a mechanism to promote the emigration of Ly6C^hi CCR2⁺ monocytes, and that TgPRF recruited inflammatory monocytes can provide an immunological benefit against an unrelated pathogen.     

Real-Time Imaging Reveals the Dynamics of Leukocyte Behaviour during Experimental Cerebral Malaria Pathogenesis

During experimental cerebral malaria (ECM) mice develop a lethal neuropathological syndrome associated with microcirculatory dysfunction and intravascular leukocyte sequestration. The precise spatio-temporal context in which the intravascular immune response unfolds is incompletely understood. We developed a 2-photon intravital microscopy (2P-IVM)-based brain-imaging model to monitor the real-time behaviour of leukocytes directly within the brain vasculature during ECM. Ly6C^hi monocytes, but not neutrophils, started to accumulate in the blood vessels of Plasmodium berghei ANKA (PbA)-infected MacGreen mice, in which myeloid cells express GFP, one to two days prior to the onset of the neurological signs (NS). A decrease in the rolling speed of monocytes, a measure of endothelial cell activation, was associated with progressive worsening of clinical symptoms. Adoptive transfer experiments with defined immune cell subsets in recombinase activating gene (RAG)-1-deficient mice showed that these changes were mediated by Plasmodium-specific CD8⁺ T lymphocytes. A critical number of CD8⁺ T effectors was required to induce disease and monocyte adherence to the vasculature. Depletion of monocytes at the onset of disease symptoms resulted in decreased lymphocyte accumulation, suggesting reciprocal effects of monocytes and T cells on their recruitment within the brain. Together, our studies define the real-time kinetics of leukocyte behaviour in the central nervous system during ECM, and reveal a significant role for Plasmodium-specific CD8⁺ T lymphocytes in regulating vascular pathology in this disease.     

Type I interferon signaling regulates Ly6C(hi) monocytes and neutrophils during acute viral pneumonia in mice

Type I interferon (IFN-I) plays a critical role in the homeostasis of hematopoietic stem cells and influences neutrophil influx to the site of inflammation. IFN-I receptor knockout (Ifnar1 ^−/−) mice develop significant defects in the infiltration of Ly6C^hi monocytes in the lung after influenza infection (A/PR/8/34, H1N1). Ly6C^hi monocytes of wild-type (WT) mice are the main producers of MCP-1 while the alternatively generated Ly6C^int monocytes of Ifnar1 ^−/− mice mainly produce KC for neutrophil influx. As a consequence, Ifnar1 ^−/− mice recruit more neutrophils after influenza infection than do WT mice. Treatment of IFNAR1 blocking antibody on the WT bone marrow (BM) cells in vitro failed to differentiate into Ly6C^hi monocytes. By using BM chimeric mice (WT BM into Ifnar1 ^−/− and vice versa), we confirmed that IFN-I signaling in hematopoietic cells is required for the generation of Ly6C^hi monocytes. Of note, WT BM reconstituted Ifnar1 ^−/− chimeric mice with increased numbers of Ly6C^hi monocytes survived longer than influenza-infected Ifnar1 ^−/− mice. In contrast, WT mice that received Ifnar1 ^−/− BM cells with alternative Ly6C^int monocytes and increased numbers of neutrophils exhibited higher mortality rates than WT mice given WT BM cells. Collectively, these data suggest that IFN-I contributes to resistance of influenza infection by control of monocytes and neutrophils in the lung.     

Ly6Chigh Monocytes Become Alternatively Activated Macrophages in Schistosome Granulomas with Help from CD4+ Cells

Alternatively activated macrophages (AAM) that accumulate during chronic T helper 2 inflammatory conditions may arise through proliferation of resident macrophages or recruitment of monocyte-derived cells. Liver granulomas that form around eggs of the helminth parasite Schistosoma mansoni require AAM to limit tissue damage. Here, we characterized monocyte and macrophage dynamics in the livers of infected CX3CR1^GFP/+ mice. CX₃CR1-GFP⁺ monocytes and macrophages accumulated around eggs and in granulomas during infection and upregulated PD-L2 expression, indicating differentiation into AAM. Intravital imaging of CX₃CR1-GFP⁺ Ly6C^low monocytes revealed alterations in patrolling behavior including arrest around eggs that were not encased in granulomas. Differential labeling of CX₃CR1-GFP⁺ cells in the blood and the tissue showed CD4⁺ T cell dependent accumulation of PD-L2⁺ CX₃CR1-GFP⁺ AAM in the tissues as granulomas form. By adoptive transfer of Ly6C^high and Ly6C^low monocytes into infected mice, we found that AAM originate primarily from transferred Ly6C^high monocytes, but that these cells may transition through a Ly6C^low state and adopt patrolling behavior in the vasculature. Thus, during chronic helminth infection AAM can arise from recruited Ly6C^high monocytes via help from CD4⁺ T cells.     

Selective chemokine receptor usage by central nervous system myeloid cells in CCR2-red fluorescent protein knock-in mice

Background

Monocyte subpopulations distinguished by differential expression of chemokine receptors CCR2 and CX3CR1 are difficult to track in vivo, partly due to lack of CCR2 reagents.

Methodology/Principal Findings

We created CCR2-red fluorescent protein (RFP) knock-in mice and crossed them with CX3CR1-GFP mice to investigate monocyte subset trafficking. In mice with experimental autoimmune encephalomyelitis, CCR2 was critical for efficient intrathecal accumulation and localization of Ly6C^hi/CCR2^hi monocytes. Surprisingly, neutrophils, not Ly6C^lo monocytes, largely replaced Ly6C^hi cells in the central nervous system of these mice. CCR2-RFP expression allowed the first unequivocal distinction between infiltrating monocytes/macrophages from resident microglia.

Conclusion/Significance

These results refine the concept of monocyte subsets, provide mechanistic insight about monocyte entry into the central nervous system, and present a novel model for imaging and quantifying inflammatory myeloid populations.     

Ly6Chi Monocyte Recruitment Is Responsible for Th2 Associated Host-Protective Macrophage Accumulation in Liver Inflammation due to Schistosomiasis

Accumulation of M2 macrophages in the liver, within the context of a strong Th2 response, is a hallmark of infection with the parasitic helminth, Schistosoma mansoni, but the origin of these cells is unclear. To explore this, we examined the relatedness of macrophages to monocytes in this setting. Our data show that both monocyte-derived and resident macrophages are engaged in the response to infection. Infection caused CCR2-dependent increases in numbers of Ly6C^hi monocytes in blood and liver and of CX₃CR1⁺ macrophages in diseased liver. Ly6C^hi monocytes recovered from liver had the potential to differentiate into macrophages when cultured with M-CSF. Using pulse chase BrdU labeling, we found that most hepatic macrophages in infected mice arose from monocytes. Consistent with this, deletion of monocytes led to the loss of a subpopulation of hepatic CD11c^hi macrophages that was present in infected but not naïve mice. This was accompanied by a reduction in the size of egg-associated granulomas and significantly exacerbated disease. In addition to the involvement of monocytes and monocyte-derived macrophages in hepatic inflammation due to infection, we observed increased incorporation of BrdU and expression of Ki67 and MHC II in resident macrophages, indicating that these cells are participating in the response. Expression of both M2 and M1 marker genes was increased in liver from infected vs. naive mice. The M2 fingerprint in the liver was not accounted for by a single cell type, but rather reflected expression of M2 genes by various cells including macrophages, neutrophils, eosinophils and monocytes. Our data point to monocyte recruitment as the dominant process for increasing macrophage cell numbers in the liver during schistosomiasis.     

Extraintestinal Helminth Infection Reduces the Development of Colitis-Associated Tumorigenesis

Colitis-associated colorectal cancer (CAC) is one of the most common cancers and is closely related to chronic or deregulated inflammation. Helminthic infections can modulate inflammatory responses in some diseases, but their immunomodulatory role during cancer development remains completely unknown. We have analyzed the role of Taenia crassiceps-induced anti-inflammatory response in determining the outcome of CAC. We show that extraintestinal T. crassiceps infection in CAC mice inhibited colonic inflammatory responses and tumor formation and prevented goblet cell loss. There was also increased expression of IL-4 and alternatively activated macrophages markers in colonic tissue and negative immunomodulation of pro-inflammatory cytokine expression. In addition, T. crassiceps infection prevented the upregulation of β-catenin and CXCR2 expression observed in the CAC mice, which are both markers associated with CAC-tumorigenesis, and reduced the numbers of circulating and colonic CD11b⁺Ly6C^hiCCR2⁺ monocytes. Thus, immunomodulatory activities induced by helminth infections may have a role in the progression of CAC.     

Influenza A virus elicits peri-vascular adipose tissue inflammation and vascular dysfunction of the aorta in pregnant mice

Influenza A virus (IAV) infection during pregnancy initiates significant aortic endothelial and vascular smooth muscle dysfunction, with inflammation and T cell activation, but the details of the mechanism are yet to be clearly defined. Here we demonstrate that IAV disseminates preferentially into the perivascular adipose tissue (PVAT) of the aorta in mice. IAV mRNA levels in the PVAT increased at 1–3 days post infection (d.p.i) with the levels being ~4–8 fold higher compared with the vessel wall. IAV infection also increased Ly6C^low patrolling monocytes and Ly6C^high pro-inflammatory monocytes in the vessel wall at 3 d.p.i., which was then followed by a greater homing of these monocytes into the PVAT at 6 d.p.i. The vascular immune phenotype was characteristic of a “vascular storm”- like response, with increases in neutrophils, pro-inflammatory cytokines and oxidative stress markers in the PVAT and arterial wall, which was associated with an impairment in endothelium-dependent relaxation to acetylcholine. IAV also triggered a PVAT compartmentalised elevation in CD4⁺ and CD8⁺ activated T cells. In conclusion, the PVAT of the aorta is a niche that supports IAV dissemination and a site for perpetuating a profound innate inflammatory and adaptive T cell response. The manifestation of this inflammatory response in the PVAT following IAV infection may be central to the genesis of cardiovascular complications arising during pregnancy.     

TNF Drives Monocyte Dysfunction with Age and Results in Impaired Anti-pneumococcal Immunity

Monocyte phenotype and output changes with age, but why this occurs and how it impacts anti-bacterial immunity are not clear. We found that, in both humans and mice, circulating monocyte phenotype and function was altered with age due to increasing levels of TNF in the circulation that occur as part of the aging process. Ly6C⁺ monocytes from old (18–22 mo) mice and CD14⁺CD16⁺ intermediate/inflammatory monocytes from older adults also contributed to this “age-associated inflammation” as they produced more of the inflammatory cytokines IL6 and TNF in the steady state and when stimulated with bacterial products. Using an aged mouse model of pneumococcal colonization we found that chronic exposure to TNF with age altered the maturity of circulating monocytes, as measured by F4/80 expression, and this decrease in monocyte maturation was directly linked to susceptibility to infection. Ly6C⁺ monocytes from old mice had higher levels of CCR2 expression, which promoted premature egress from the bone marrow when challenged with Streptococcus pneumoniae. Although Ly6C⁺ monocyte recruitment and TNF levels in the blood and nasopharnyx were higher in old mice during S. pneumoniae colonization, bacterial clearance was impaired. Counterintuitively, elevated TNF and excessive monocyte recruitment in old mice contributed to impaired anti-pneumococcal immunity since bacterial clearance was improved upon pharmacological reduction of TNF or Ly6C⁺ monocytes, which were the major producers of TNF. Thus, with age TNF impairs inflammatory monocyte development, function and promotes premature egress, which contribute to systemic inflammation and is ultimately detrimental to anti-pneumococcal immunity.     

Functional Interplay between Type I and II Interferons Is Essential to Limit Influenza A Virus-Induced Tissue Inflammation

Host control of influenza A virus (IAV) is associated with exuberant pulmonary inflammation characterized by the influx of myeloid cells and production of proinflammatory cytokines including interferons (IFNs). It is unclear, however, how the immune system clears the virus without causing lethal immunopathology. Here, we demonstrate that in addition to its known anti-viral activity, STAT1 signaling coordinates host inflammation during IAV infection in mice. This regulatory mechanism is dependent on both type I IFN and IFN-γ receptor signaling and, importantly, requires the functional interplay between the two pathways. The protective function of type I IFNs is associated with not only the recruitment of classical inflammatory Ly6C^hi monocytes into IAV-infected lungs, but also the prevention of excessive monocyte activation by IFN-γ. Unexpectedly, type I IFNs preferentially regulate IFN-γ signaling in Ly6C^lo rather than inflammatory Ly6C^hi mononuclear cell populations. In the absence of type I IFN signaling, Ly6C^lo monocytes/macrophages, become phenotypically and functionally more proinflammatory than Ly6C^hi cells, revealing an unanticipated function of the Ly6C^lo mononuclear cell subset in tissue inflammation. In addition, we show that type I IFNs employ distinct mechanisms to regulate monocyte and neutrophil trafficking. Type I IFN signaling is necessary, but not sufficient, for preventing neutrophil recruitment into the lungs of IAV-infected mice. Instead, the cooperation of type I IFNs and lymphocyte-produced IFN-γ is required to regulate the tissue neutrophilic response to IAV. Our study demonstrates that IFN interplay links innate and adaptive anti-viral immunity to orchestrate tissue inflammation and reveals an additional level of complexity for IFN-dependent regulatory mechanisms that function to prevent excessive immunopathology while preserving anti-microbial functions.     

Type I Interferons Promote Fatal Immunopathology by Regulating Inflammatory Monocytes and Neutrophils during Candida Infections

Invasive fungal infections by Candida albicans (Ca) are a frequent cause of lethal sepsis in intensive care unit patients. While a contribution of type I interferons (IFNs-I) in fungal sepsis remains unknown, these immunostimulatory cytokines mediate the lethal effects of endotoxemia and bacterial sepsis. Using a mouse model lacking a functional IFN-I receptor (Ifnar1^−/−), we demonstrate a remarkable protection against invasive Ca infections. We discover a mechanism whereby IFN-I signaling controls the recruitment of inflammatory myeloid cells, including Ly6C^hi monocytes and neutrophils, to infected kidneys by driving expression of the chemokines CCL2 and KC. Within kidneys, monocytes differentiate into inflammatory DCs but fail to functionally mature in Ifnar1^−/− mice, as demonstrated by the impaired upregulation of the key activation markers PDCA1 and iNOS. The increased activity of inflammatory monocytes and neutrophils results in hyper-inflammation and lethal kidney pathology. Pharmacological diminution of monocytes and neutrophils by treating mice with pioglitazone, a synthetic agonist of the nuclear receptor peroxisome proliferator-activated receptor-γ (PPAR-γ), strongly reduces renal immunopathology during Ca infection and improves mouse survival. Taken together, our data connect for the first time the sepsis-promoting functions of IFNs-I to the CCL2-mediated recruitment and the activation of inflammatory monocytes/DCs with high host-destructing potency. Moreover, our data demonstrate a therapeutic relevance of PPAR-γ agonists for microbial infectious diseases where inflammatory myeloid cells may contribute to fatal tissue damage.     

CD8+ T cells and IFN-γ mediate the time-dependent accumulation of infected red blood cells in deep organs during experimental cerebral malaria

Background

Infection with Plasmodium berghei ANKA (PbA) in susceptible mice induces a syndrome called experimental cerebral malaria (ECM) with severe pathologies occurring in various mouse organs. Immune mediators such as T cells or cytokines have been implicated in the pathogenesis of ECM. Red blood cells infected with PbA parasites have been shown to accumulate in the brain and other tissues during infection. This accumulation is thought to be involved in PbA–induced pathologies, which mechanisms are poorly understood.

Methods and Findings

Using transgenic PbA parasites expressing the luciferase protein, we have assessed by real-time in vivo imaging the dynamic and temporal contribution of different immune factors in infected red blood cell (IRBC) accumulation and distribution in different organs during PbA infection. Using deficient mice or depleting antibodies, we observed that CD8⁺ T cells and IFN-γ drive the rapid increase in total parasite biomass and accumulation of IRBC in the brain and in different organs 6–12 days post-infection, at a time when mice develop ECM. Other cells types like CD4⁺ T cells, monocytes or neutrophils or cytokines such as IL-12 and TNF-α did not influence the early increase of total parasite biomass and IRBC accumulation in different organs.

Conclusions

CD8⁺ T cells and IFN-γ are the major immune mediators controlling the time-dependent accumulation of P. berghei-infected red blood cells in tissues.     

Persistent Salmonellosis Causes Pancreatitis in a Murine Model of Infection

Pancreatitis, a known risk factor for the development of pancreatic ductal adenocarcinoma, is a serious, widespread medical condition usually caused by alcohol abuse or gallstone-mediated ductal obstruction. However, many cases of pancreatitis are of an unknown etiology. Pancreatitis has been linked to bacterial infection, but causality has yet to be established. Here, we found that persistent infection of mice with the bacterial pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) was sufficient to induce pancreatitis reminiscent of the human disease. Specifically, we found that pancreatitis induced by persistent S. Typhimurium infection was characterized by a loss of pancreatic acinar cells, acinar-to-ductal metaplasia, fibrosis and accumulation of inflammatory cells, including CD11b⁺ F4/80⁺, CD11b⁺ Ly6C^int Ly6G⁺ and CD11b⁺ Ly6C^hi Ly6G⁻ cells. Furthermore, we found that S. Typhimurium colonized and persisted in the pancreas, associated with pancreatic acinar cells in vivo, and could invade cultured pancreatic acinar cells in vitro. Thus, persistent infection of mice with S. Typhimurium may serve as a useful model for the study of pancreatitis as it relates to bacterial infection. Increased knowledge of how pathogenic bacteria can cause pancreatitis will provide a more integrated picture of the etiology of the disease and could lead to the development of new therapeutic approaches for treatment and prevention of pancreatitis and pancreatic ductal adenocarcinoma.     

TNF‐α has both stimulatory and inhibitory effects on mouse monocyte‐derived osteoclastogenesis

Phenotypically different osteoclasts may be generated from different subsets of precursors. To what extent the formation of these osteoclasts is influenced or mediated by the inflammatory cytokine TNF‐α, is unknown and was investigated in this study. The osteoclast precursors early blasts (CD31^hiLy‐6C^?), myeloid blasts (CD31⁺Ly‐6C⁺), and monocytes (CD31^?Ly‐6C^hi) were sorted from mouse bone marrow using flow cytometry and cultured with M‐CSF and RANKL, with or without TNF‐α. Surprisingly, TNF‐α prevented the differentiation of TRAcP⁺ osteoclasts generated from monocytes on plastic; an effect not seen with early blasts and myeloid blasts. This inhibitory effect could not be prevented by other cytokines such as IL‐1β or IL‐6. When monocytes were pre‐cultured with M‐CSF and RANKL followed by exposure to TNF‐α, a stimulatory effect was found. TNF‐α also stimulated monocytes’ osteoclastogenesis when the cells were seeded on bone. Gene expression analysis showed that when TNF‐α was added to monocytes cultured on plastic, RANK, NFATc1, and TRAcP were significantly down‐regulated while TNF‐αR1 and TNF‐αR2 were up‐regulated. FACS analysis showed a decreased uptake of fluorescently labeled RANKL in monocyte cultures in the presence of TNF‐α, indicating an altered ratio of bound‐RANK/unbound‐RANK. Our findings suggest a diverse role of TNF‐α on monocytes’ osteoclastogenesis: it affects the RANK‐signaling pathway therefore inhibits osteoclastogenesis when added at the onset of monocyte culturing. This can be prevented when monocytes were pre‐cultured with M‐CSF and RANKL, which ensures the binding of RANKL to RANK. This could be a mechanism to prevent unfavorable monocyte‐derived osteoclast formation away from the bone.

     

Monocyte Recruitment to the Dermis and Differentiation to Dendritic Cells Increases the Targets for Dengue Virus Replication

Dengue virus (DENV) causes the most prevalent arthropod-borne viral disease in humans. Although Aedes mosquitoes transmit DENV when probing for blood in the skin, no information exists on DENV infection and immune response in the dermis, where the blood vessels are found. DENV suppresses the interferon response, replicates, and causes disease in humans but not wild-type mice. Here, we used mice lacking the interferon-α/β receptor (Ifnar ^–/–), which had normal cell populations in the skin and were susceptible to intradermal DENV infection, to investigate the dynamics of early DENV infection of immune cells in the skin. CD103⁺ classical dendritic cells (cDCs), Ly6C^– CD11b⁺ cDCs, and macrophages in the steady-state dermis were initial targets of DENV infection 12-24 hours post-inoculation but then decreased in frequency. We demonstrated recruitment of adoptively-transferred Ly6C^high monocytes from wild-type and Ifnar ^–/– origin to the DENV-infected dermis and differentiation to Ly6C⁺ CD11b⁺ monocyte-derived DCs (moDCs), which became DENV-infected after 48 hours, and were then the major targets for virus replication. Ly6C^high monocytes that entered the DENV-infected dermis expressed chemokine receptor CCR2, likely mediating recruitment. Further, we show that ∼100-fold more hematopoietic cells in the dermis were DENV-infected compared to Langerhans cells in the epidermis. Overall, these results identify the dermis as the main site of early DENV replication and show that DENV infection in the skin occurs in two waves: initial infection of resident cDCs and macrophages, followed by infection of monocytes and moDCs that are recruited to the dermis. Our study reveals a novel viral strategy of exploiting monocyte recruitment to increase the number of targets for infection at the site of invasion in the skin and highlights the skin as a potential site for therapeutic action or intradermal vaccination.     

CX3CR1 Signaling on Monocytes Is Dispensable after Intracerebral Hemorrhage

Intracerebral hemorrhage is a subset of stroke for which there is no specific treatment. The Ly6C^hi CCR2⁺ monocytes have been shown to contribute to acute injury after intracerebral hemorrhage. The other murine monocyte subset expresses CX3CR1 and lower Ly6C levels, and contributes to repair in other disease models. We hypothesized that the Ly6C^lo CX3CR1⁺ monocytes would contribute to recovery after intracerebral hemorrhage. Intracerebral hemorrhage was modeled by blood injection in WT and CX3CR1-null bone marrow chimeras. Neurological outcomes and leukocyte recruitment were quantified at various time points. Functional outcomes were equal at 1, 3, 7, and 14 days after intracerebral hemorrhage in both genotypes. No differences were observed in leukocyte recruitment between genotypes on either 3 or 7 days after intracerebral hemorrhage. A few hundred Ly6C^lo monocytes were found in the ipsilateral hemisphere in each genotype and they did not change over time. Peripherally derived CX3CR1⁺ monocytes were observed in the perihematomal brain 7 and 14 days after intracerebral hemorrhage. Our data suggests CX3CR1 signaling on monocytes does not play an influential role in acute injury or functional recovery after intracerebral hemorrhage and therefore CX3CR1 is not a therapeutic target to improve outcome after intracerebral hemorrhage.     

Neutralization of pro‐inflammatory monocytes by targeting TLR2 dimerization ameliorates colitis

Monocytes have emerged as critical driving force of acute inflammation. Here, we show that inhibition of Toll‐like receptor 2(TLR2) dimerization by a TLR2 transmembrane peptide (TLR2‐p) ameliorated DSS‐induced colitis by interfering specifically with the activation of Ly6C⁺ monocytes without affecting their recruitment to the colon. We report that TLR2‐p directly interacts with TLR2 within the membrane, leading to inhibition of TLR2–TLR6/1 assembly induced by natural ligands. This was associated with decreased levels of extracellular signal‐regulated kinases (ERK) signaling and reduced secretion of pro‐inflammatory cytokines, such as interleukin (IL)‐6, IL‐23, IL‐12, and IL‐1β. Altogether, our study provides insights into the essential role of TLR2 dimerization in the activation of pathogenic pro‐inflammatory Ly6C^hi monocytes and suggests that inhibition of this aggregation by TLR2‐p might have therapeutic potential in the treatment of acute gut inflammation.     

Chronic Parasitic Infection Maintains High Frequencies of Short-Lived Ly6C+CD4+ Effector T Cells That Are Required for Protection against Re-infection

In contrast to the ability of long-lived CD8⁺ memory T cells to mediate protection against systemic viral infections, the relationship between CD4⁺ T cell memory and acquired resistance against infectious pathogens remains poorly defined. This is especially true for T helper 1 (Th1) concomitant immunity, in which protection against reinfection coincides with a persisting primary infection. In these situations, pre-existing effector CD4 T cells generated by ongoing chronic infection, not memory cells, may be essential for protection against reinfection. We present a systematic study of the tissue homing properties, functionality, and life span of subsets of memory and effector CD4 T cells activated in the setting of chronic Leishmania major infection in resistant C57Bl/6 mice. We found that pre-existing, CD44⁺CD62L⁻T-bet⁺Ly6C⁺ effector (T_EFF) cells that are short-lived in the absence of infection and are not derived from memory cells reactivated by secondary challenge, mediate concomitant immunity. Upon adoptive transfer and challenge, non-dividing Ly6C⁺ T_EFF cells preferentially homed to the skin, released IFN-γ, and conferred protection as compared to CD44⁺CD62L⁻Ly6C⁻ effector memory or CD44⁺CD62L⁺Ly6C⁻ central memory cells. During chronic infection, Ly6C⁺ T_EFF cells were maintained at high frequencies via reactivation of T_CM and the T_EFF themselves. The lack of effective vaccines for many chronic diseases may be because protection against infectious challenge requires the maintenance of pre-existing T_EFF cells, and is therefore not amenable to conventional, memory inducing, vaccination strategies.     

Successful colon cancer eradication after chemoimmunotherapy is associated with profound phenotypic change of intratumoral myeloid cells

IL-12 is a potent immunostimulatory cytokine, but its impact as an antitumor drug in clinical practice is limited. Upsurge of regulatory T cells (Treg) in the tumor milieu has been proposed to limit the efficacy of the treatment. In this paper, two drugs (cyclophosphamide [CPA] and anti-CD25 mAb) widely used to eliminate Treg were used in an attempt to enhance the antitumor effect of IL-12 gene therapy. Both anti-CD25 and CPA combined with IL-12 were able to deplete intratumoral Treg and myeloid-derived suppressor cells (MDSC), but only IL-12 plus CPA achieved significant antitumor activity in mice with large established s.c. colon carcinoma. This therapeutic effect was associated with the emergence of a heterogeneous population of myeloid cells within the tumor, termed inflammatory myeloid cells (IMC), composed of Ly6C(high)Ly6G(low) inflammatory monocytes and Ly6G(high)Ly6C(+) neutrophils. IMC showed a distinctive pattern of cytokine/chemokine production, and in contrast to MDSC, they did not induce conversion of naive CD4(+) T cells into Treg. The appearance of IMC coincided with intense tumor infiltration by effector T cells, which was abrogated by elimination of IMC by anti-Gr1 mAb, a maneuver that abolished the antitumor effect of the therapy. Therefore, the combination of IL-12 and CPA eliminates intratumoral Treg and MDSC, while it induces the appearance of IMC within the tumor microenvironment. The latter effect is essential to facilitate effector T cell infiltration and subsequent tumor elimination.