WSX-1 Signalling Inhibits CD4+ T Cell Migration to the Liver during Malaria Infection by Repressing Chemokine-Independent Pathways

IL-27 is an important and non-redundant regulator of effector T cell accumulation in non-lymphoid tissues during infection. Using malaria as a model systemic pro-inflammatory infection, we demonstrate that the aberrant accumulation of CD4⁺ T cells in the liver of infected IL27R^−/− (WSX-1^−/−) mice is a result of differences in cellular recruitment, rather than changes in T cell proliferation or cell death. We show that IL-27 both inhibits the migratory capacity of infection-derived CD4⁺ T cells towards infection-derived liver cells, but also suppresses the production of soluble liver-derived mediator(s) that direct CD4⁺ T cell movement towards the inflamed tissue. Although CCL4 and CCL5 expression was higher in livers of infected WSX-1^−/− mice than infected WT mice, and hepatic CD4⁺ T cells from WSX-1^−/− mice expressed higher levels of CCR5 than cells from WT mice, migration of CD4⁺ T cells to the liver of WSX-1^−/− mice during infection was not controlled by chemokine (R) signalling. However, anti-IL-12p40 treatment reduced migration of CD4⁺ T cells towards infection-derived liver cells, primarily by abrogating the hepatotropic migratory capacity of T cells, rather than diminishing soluble tissue-derived migratory signals. These results indicate that IL-27R signalling restricts CD4⁺ T cell accumulation within the liver during infection primarily by suppressing T cell chemotaxis, which may be linked to its capacity to repress Th1 differentiation, as well as by inhibiting the production of soluble, tissue-derived chemotaxins.     

Deletion of IL-33R (ST2) Abrogates Resistance to EAE in BALB/C Mice by Enhancing Polarization of APC to Inflammatory Phenotype

The administration of interleukin 33 and deletion of IL-33 receptor, ST2 molecule, affects the induction of autoimmunity in different experimental models of human autoimmune diseases. The aim of this study was to analyze the effect of ST2 deletion on the induction of experimental autoimmune encephalomyelitis (EAE) in resistant BALB/c mice. Mice were immunized with MOG_35–55 peptide or disease was induced by passive transfer of encephalitogenic singenic cells and EAE was clinically and histologically evaluated. Expression of intracellular inflammatory cytokines, markers of activation and chemokine receptors on lymphoid tissue and CNS infiltrating mononuclear cells was analyzed by flow cytometry. We report here that deletion of ST2^−/− molecule abrogates resistance of BALB/c mice to EAE induction based on clinical and histopathological findings. Brain and spinal cord infiltrates of ST2^−/− mice had significantly higher number of CD4⁺ T lymphocytes containing inflammatory cytokines compared to BALB/c WT mice. Adoptive transfer of ST2^−/− primed lymphocytes induced clinical signs of the disease in ST2^−/− as well as in WT mice. MOG_35–55 restimulated ST2^−/− CD4⁺ cells as well as ex vivo analyzed lymph node cells had higher expression of T-bet and IL-17, IFN-γ, TNF-α and GM-CSF in comparison with WT CD4⁺ cells. ST2^−/− mice had higher percentages of CD4⁺ cells expressing chemokine receptors important for migration to CNS in comparison with WT CD4⁺ cells. Draining lymph nodes of ST2^−/− mice contained higher percentage of CD11c⁺CD11b⁺CD8⁻ cells containing inflammatory cytokines IL-6 and IL-12 with higher expression of activation markers. Transfer of ST2^−/− but not WT dendritic cells induced EAE in MOG_35–55 immunized WT mice. Our results indicate that ST2 deficiency attenuates inherent resistance of BALB/c mice to EAE induction by enhancing differentiation of proinflammatory antigen presenting cells and consecutive differentiation of encephalitogenic T cells in the draining lymph node rather than affecting their action in the target tissue.     

IL-23 Induces Atopic Dermatitis-Like Inflammation Instead of Psoriasis-Like Inflammation in CCR2-Deficient Mice

Psoriasis is an immune-mediated chronic inflammatory skin disease, characterized by epidermal hyperplasia and infiltration of leukocytes into the dermis and epidermis. IL-23 is expressed in psoriatic skin, and IL-23 injected into the skin of mice produces IL-22-dependent dermal inflammation and acanthosis. The chemokine receptor CCR2 has been implicated in the pathogenesis of several inflammatory diseases, including psoriasis. CCR2-positive cells and the CCR2 ligand, CCL2 are abundant in psoriatic lesions. To examine the requirement of CCR2 in the development of IL-23-induced cutaneous inflammation, we injected the ears of wild-type (WT) and CCR2-deficient (CCR2^−/−) mice with IL-23. CCR2^−/− mice had increased ear swelling and epidermal thickening, which was correlated with increased cutaneous IL-4 levels and increased numbers of eosinophils within the skin. In addition, TSLP, a cytokine known to promote and amplify T helper cell type 2 (Th2) immune responses, was also increased within the inflamed skin of CCR2^−/− mice. Our data suggest that increased levels of TSLP in CCR2^−/− mice may contribute to the propensity of these mice to develop increased Th2-type immune responses.     

Role of the monocyte chemoattractant protein-1/C-C chemokine receptor 2 signaling pathway in transient receptor potential vanilloid type 1 ablation-induced renal injury in salt-sensitive hypertension

Our recent studies indicate that the transient receptor potential vanilloid type 1 (TRPV1) channel may act as a potential regulator of monocyte/macrophage recruitment to reduce renal injury in salt-sensitive hypertension. This study tests the hypothesis that deletion of TRPV1 exaggerates salt-sensitive hypertension-induced renal injury due to enhanced inflammatory responses via monocyte chemoattractant protein-1 (MCP-1)/C-C chemokine receptor 2 (CCR2)-dependent pathways. Wild type (WT) and TRPV1-null mutant (TRPV1^−/−) mice were subjected to uninephrectomy and deoxycorticosterone acetate (DOCA)-salt treatment for four weeks with or without the selective CCR2 antagonist, RS504393. DOCA-salt treatment increased systolic blood pressure (SBP) to the same degree in both strains, but increased urinary excretion of albumin and 8-isoprostane and decreased creatinine clearance with greater magnitude in TRPV1^−/− mice compared to WT mice. DOCA-salt treatment also caused renal glomerulosclerosis, tubulointerstitial injury, collagen deposition, monocyte/macrophage infiltration, proinflammatory cytokine and chemokine production, and NF-κB activation in greater degree in TRPV1^−/− mice compared to WT mice. Blockade of the CCR2 with RS504393 (4 mg/kg/day) had no effect on SBP in DOCA-salt-treated WT or TRPV1^−/− mice compared to their respective controls. However, treatment with RS504393 ameliorated renal dysfunction and morphological damage, and prevented the increase in monocyte/macrophage infiltration, cytokine/chemokine production, and NF-κB activity in both DOCA-salt hypertensive strains with a greater effect in DOCA-salt-treated TRPV1^−/− mice compared to DOCA-salt-treated WT mice. No differences in CCR2 protein expression in kidney were found between DOCA-salt-treated WT and TRPV1^−/− mice with or without RS504393 treatment. Our studies for the first time indicate that deletion of TRPV1 aggravated renal injury in salt-sensitive hypertension via enhancing MCP-1/CCR2 signaling-dependent inflammatory responses.     

MCP/CCR2 Signaling Is Essential for Recruitment of Mesenchymal Progenitor Cells during the Early Phase of Fracture Healing

Objective

The purpose of this study was to investigate chemokine profiles and their functional roles in the early phase of fracture healing in mouse models.

Methods

The expression profiles of chemokines were examined during fracture healing in wild-type (WT) mice using a polymerase chain reaction array and histological staining. The functional effect of monocyte chemotactic protein-1 (MCP-1) on primary mouse bone marrow stromal cells (mBMSCs) was evaluated using an in vitro migration assay. MCP-1^−/− and C-C chemokine receptor 2 (CCR2)^−/− mice were fractured and evaluated by histological staining and micro-computed tomography (micro-CT). RS102895, an antagonist of CCR2, was continuously administered in WT mice before or after rib fracture and evaluated by histological staining and micro-CT. Bone graft exchange models were created in WT and MCP-1^−/− mice and were evaluated by histological staining and micro-CT.

Results

MCP-1 and MCP-3 expression in the early phase of fracture healing were up-regulated, and high levels of MCP-1 and MCP-3 protein expression observed in the periosteum and endosteum in the same period. MCP-1, but not MCP-3, increased migration of mBMSCs in a dose-dependent manner. Fracture healing in MCP-1^−/− and CCR2^−/− mice was delayed compared with WT mice on day 21. Administration of RS102895 in the early, but not in the late phase, caused delayed fracture healing. Transplantation of WT-derived graft into host MCP-1^−/− mice significantly increased new bone formation in the bone graft exchange models. Furthermore, marked induction of MCP-1 expression in the periosteum and endosteum was observed around the WT-derived graft in the host MCP-1^−/− mouse. Conversely, transplantation of MCP-1^−/− mouse-derived grafts into host WT mice markedly decreased new bone formation.

Conclusions

MCP-1/CCR2 signaling in the periosteum and endosteum is essential for the recruitment of mesenchymal progenitor cells in the early phase of fracture healing.     

CD22 Is Required for Protection against West Nile Virus Infection

West Nile virus (WNV) is a RNA virus of the family Flaviviridae and the leading cause of mosquito-borne encephalitis in the United States. Humoral immunity is essential for protection against WNV infection; however, the requirements for initiating effective antibody responses against WNV infection are still unclear. CD22 (Siglec-2) is expressed on B cells and regulates B cell receptor signaling, cell survival, proliferation, and antibody production. In this study, we investigated how CD22 contributes to protection against WNV infection and found that CD22 knockout (Cd22^−/−) mice were highly susceptible to WNV infection and had increased viral loads in the serum and central nervous system (CNS) compared to wild-type (WT) mice. This was not due to a defect in humoral immunity, as Cd22^−/− mice had normal WNV-specific antibody responses. However, Cd22^−/− mice had decreased WNV-specific CD8⁺ T cell responses compared to those of WT mice. These defects were not simply due to reduced cytotoxic activity or increased cell death but, rather, were associated with decreased lymphocyte migration into the draining lymph nodes (dLNs) of infected Cd22^−/− mice. Cd22^−/− mice had reduced production of the chemokine CCL3 in the dLNs after infection, suggesting that CD22 affects chemotaxis via controlling chemokine production. CD22 was not restricted to B cells but was also expressed on a subset of splenic DCIR2⁺ dendritic cells that rapidly expand early after WNV infection. Thus, CD22 plays an essential role in controlling WNV infection by governing cell migration and CD8⁺ T cell responses.     

Potential Roles of CCR5+ CCR6+ Dendritic Cells Induced by Nasal Ovalbumin plus Flt3 Ligand Expressing Adenovirus for Mucosal IgA Responses

We assessed the role of CCR5⁺/CCR6⁺/CD11b⁺/CD11c⁺ dendritic cells (DCs) for induction of ovalbumin (OVA)-specific antibody (Ab) responses following mucosal immunization. Mice given nasal OVA plus an adenovirus expressing Flt3 ligand (Ad-FL) showed early expansion of CCR5⁺/CCR6⁺/CD11b⁺/CD11c⁺ DCs in nasopharyngeal-associated lymphoid tissue (NALT) and cervical lymph nodes (CLNs). Subsequently, this DC subset became resident in submandibular glands (SMGs) and nasal passages (NPs) in response to high levels of CCR-ligands produced in these tissues. CD11b⁺/CD11c⁺ DCs were markedly decreased in both CCR5^−/− and CCR6^−/− mice. Chimera mice reconstituted with bone marrow cells from CD11c-diphtheria toxin receptor (CD11c-DTR) and CCR5^−/− or CD11c-DTR and CCR6^−/− mice given nasal OVA plus Ad-FL had elevated plasma IgG, but reduced IgA as well as low anti-OVA secretory IgA (SIgA )Ab responses in saliva and nasal washes. These results suggest that CCR5⁺CCR6⁺ DCs play an important role in the induction of Ag-specific SIgA Ab responses.     

P47phox?/? Mice Are Compromised in Expansion and Activation of CD8+ T Cells and Susceptible to Trypanosoma cruzi Infection

Macrophage activation of NAD(P)H oxidase (NOX2) and reactive oxygen species (ROS) is suggested to kill Trypanosoma cruzi that causes Chagas disease. However, the role of NOX2 in generation of protective immunity and whether these mechanisms are deregulated in the event of NOX2 deficiency are not known, and examined in this study. Our data showed that C57BL/6 p47^phox−/− mice (lack NOX2 activity), as compared to wild-type (WT) mice, succumbed within 30 days post-infection (pi) to low doses of T. cruzi and exhibited inability to control tissue parasites. P47^phox−/− bone-marrow and splenic monocytes were not compromised in maturation, phagocytosis and parasite uptake capacity. The deficiency of NOX2 mediated ROS was compensated by higher level of inducible nitric oxide synthase (iNOS) expression, and nitric oxide and inflammatory cytokine (TNF-α, IFN-γ, IL-1β) release by p47^phox−/− macrophages as compared to that noted in WT controls infected by T. cruzi. Splenic activation of Th1 CD4⁺T cells and tissue infiltration of immune cells in T. cruzi infected p47^phox−/− mice were comparable to that noted in infected control mice. However, generation and activation of type 1 CD8⁺T cells was severely compromised in p47^phox−/− mice. In comparison, WT mice exhibited a robust T. cruzi-specific CD8⁺T cell response with type 1 (IFN-γ+TNF-α>IL-4+IL-10), cytolytic effector (CD8⁺CD107a⁺IFN-γ⁺) phenotype. We conclude that NOX2/ROS activity in macrophages signals the development of antigen-specific CD8⁺T cell response. In the event of NOX2 deficiency, a compromised CD8⁺T cell response is generated, leading to increased parasite burden, tissue pathogenesis and mortality in chagasic mice.     

Downregulation of Key Early Events in the Mobilization of Antigen-bearing Dendritic Cells by Leukocyte Immunoglobulin-like Receptor B4 in a Mouse Model of Allergic Pulmonary Inflammation

Leukocyte Immunoglobulin-like Receptor B4 (LILRB4) null mice have an exacerbated T helper cell type 2 (Th2) immune response and pulmonary inflammation compared with Lilrb4^+/+ animals when sensitized intranasally with ovalbumin (OVA) and low-dose lipopolysaccharide (LPS) followed by challenge with OVA. Moreover, OVA-challenged Lilrb4 ^−/− mice exhibit greater migration of antigen (Ag)-bearing dendritic cells (DCs) to lymph nodes and accumulation of interleukin 4- and interleukin 5-producing lymph node lymphocytes. The main objective of this study was to determine how the absence of LILRB4 leads to a greater number of DCs in the lymph nodes of Ag-challenged mice and increased lung Th2 inflammation. Mice were sensitized intranasally with PBS alone or containing OVA and LPS; additional cohorts were subsequently challenged with OVA. Expression of chemokine (C-C motif) ligand 21 (CCL21) in the lung was assessed immunohistologically. OVA ingestion and expression of LILRB4 and chemokine (C-C motif) receptor 7 (CCR7) were quantified by flow cytometry. Inhalation of OVA and LPS induced upregulation of LILRB4 selectively on lung Ag-bearing DCs. After sensitization and challenge, the lung lymphatic vessels of Lilrb4 ^−/− mice expressed more CCL21, a chemokine that directs the migration of DCs from peripheral tissue to draining lymph nodes, compared with Lilrb4^+/+ mice. In addition, lung DCs of challenged Lilrb4 ^−/− mice expressed more CCR7, the CCL21 receptor. The lungs of challenged Lilrb4 ^−/− mice also contained significantly greater numbers of CD4+ cells expressing interleukin-4 or interleukin-5, consistent with the greater number of Ag-bearing DCs and Th2 cells in lymph nodes and the attendant exacerbated Th2 lung pathology. Our data establish a new mechanism by which LILRB4 can downregulate the development of pathologic allergic inflammation: reduced upregulation of key molecules needed for DC migration leading to decreases in Th2 cells in lymph nodes and their target tissue.     

CCR2 and CD44 Promote Inflammatory Cell Recruitment during Fatty Liver Formation in a Lithogenic Diet Fed Mouse Model

Non-alcoholic fatty liver disease (NAFLD) is a common disease with a spectrum of presentations. The current study utilized a lithogenic diet model of NAFLD. The diet was fed to mice that are either resistant (AKR) or susceptible (BALB/c and C57BL/6) to hepatitis followed by molecular and flow cytometric analysis. Following this, a similar approach was taken in congenic mice with specific mutations in immunological genes. The initial study identified a significant and profound increase in multiple ligands for the chemokine receptor CCR2 and an increase in CD44 expression in susceptible C57BL/6 (B6) but not resistant AKR mice. Ccr2^−/− mice were completely protected from hepatitis and Cd44^−/− mice were partially protected. Despite protection from inflammation, both strains displayed similar histological steatosis scores and significant increases in serum liver enzymes. CD45⁺CD44⁺ cells bound to hyaluronic acid (HA) in diet fed B6 mice but not Cd44^−/− or Ccr2^−/− mice. Ccr2^−/− mice displayed a diminished HA binding phenotype most notably in monocytes, and CD8⁺ T-cells. In conclusion, this study demonstrates that absence of CCR2 completely and CD44 partially reduces hepatic leukocyte recruitment. These data also provide evidence that there are multiple redundant CCR2 ligands produced during hepatic lipid accumulation and describes the induction of a strong HA binding phenotype in response to LD feeding in some subsets of leukocytes from susceptible strains.     

CD8α Dendritic Cells Drive Establishment of HSV-1 Latency

It is generally accepted that CD8 T cells play the key role to maintain HSV-1 latency in trigeminal ganglia of ocularly infected mice. Yet, comparably little is known about the role of innate immunity in establishment of viral latency. In the current study, we investigated whether CD8α DCs impact HSV-1 latency by examining latency in the trigeminal ganglia (TG) of wild-type (WT) C57BL/6 versus CD8α^−/− (lack functional CD8 T cells and CD8α⁺ DCs), CD8β^−/− (have functional CD8α⁺ T cells and CD8α⁺ DCs), and β2m^−/− (lack functional CD8 T cells but have CD8α⁺ DCs) mice as well as BXH2 (have functional CD8 T cells but lack CD8α⁺ DCs) versus WT C3H (have functional CD8α T cells and CD8α⁺ DCs) mice. We also determined whether the phenotype of CD8α^−/− and BXH2 mice could be restored to that of WT mice by adoptive transfer of WT CD8⁺ T cells or bone marrow (BM) derived CD8α⁺ DCs. Our results clearly demonstrate that CD8α DCs, rather than CD8 T cells, are responsible for enhanced viral latency and recurrences.     

Preferential HIV Infection of CCR6+ Th17 Cells Is Associated with Higher Levels of Virus Receptor Expression and Lack of CCR5 Ligands

Th17 cells are enriched in the gut mucosa and play a critical role in maintenance of the mucosal barrier and host defense against extracellular bacteria and fungal infections. During chronic human immunodeficiency virus (HIV) infection, Th17 cells were more depleted compared to Th1 cells, even when the patients had low or undetectable viremia. To investigate the differential effects of HIV infection on Th17 and Th1 cells, a culture system was used in which CCR6⁺ CD4⁺ T cells were sorted from healthy human peripheral blood and activated in the presence of interleukin 1β (IL-1β) and IL-23 to drive expansion of Th17 cells while maintaining Th1 cells. HIV infection of these cultures had minimal effects on Th1 cells but caused depletion of Th17 cells. Th17 loss correlated with greater levels of virus-infected cells and cell death. In identifying cellular factors contributing to higher susceptibility of Th17 cells to HIV, we compared Th17-enriched CCR6⁺ and Th17-depleted CCR6⁻ CD4 T cell cultures and noted that Th17-enriched CCR6⁺ cells expressed higher levels of α4β7 and bound HIV envelope in an α4β7-dependent manner. The cells also had greater expression of CD4 and CXCR4, but not CCR5, than CCR6⁻ cells. Moreover, unlike Th1 cells, Th17 cells produced little CCR5 ligand, and transfection with one of the CCR5 ligands, MIP-1β (CCL4), increased their resistance against HIV. These results indicate that features unique to Th17 cells, including higher expression of HIV receptors and lack of autocrine CCR5 ligands, are associated with enhanced permissiveness of these cells to HIV.     

In Pulmonary Paracoccidioidomycosis IL-10 Deficiency Leads to Increased Immunity and Regressive Infection without Enhancing Tissue Pathology

Background

Cellular immunity is the main defense mechanism in paracoccidioidomycosis (PCM), the most important systemic mycosis in Latin America. Th1 immunity and IFN-γ activated macrophages are fundamental to immunoprotection that is antagonized by IL-10, an anti-inflammatory cytokine. Both in human and experimental PCM, several evidences indicate that the suppressive effect of IL-10 causes detrimental effects to infected hosts. Because direct studies have not been performed, this study was aimed to characterize the function of IL-10 in pulmonary PCM.

Methodology/Principal Findings

Wild type (WT) and IL-10^−/− C57BL/6 mice were used to characterize the role of IL-10 in the innate and adaptive immunity against Paracoccidioides brasiliensis (Pb) infection. We verified that Pb-infected peritoneal macrophages from IL-10^−/− mice presented higher phagocytic and fungicidal activities than WT macrophages, and these activities were associated with elevated production of IFN-γ, TNF-α, nitric oxide (NO) and MCP-1. For in vivo studies, IL-10^−/− and WT mice were i.t. infected with 1×10⁶ Pb yeasts and studied at several post-infection periods. Compared to WT mice, IL-10^−/− mice showed increased resistance to P. brasiliensis infection as determined by the progressive control of pulmonary fungal loads and total clearance of fungal cells from dissemination organs. This behavior was accompanied by enhanced delayed-type hypersensitivity reactions, precocious humoral immunity and controlled tissue pathology resulting in increased survival times. In addition, IL-10^−/− mice developed precocious T cell immunity mediated by increased numbers of lung infiltrating effector/memory CD4⁺ and CD8⁺ T cells. The inflammatory reactions and the production of Th1/Th2/Th17 cytokines were reduced at late phases of infection, paralleling the regressive infection of IL-10^−/− mice.

Conclusions/Significance

Our work demonstrates for the first time that IL-10 plays a detrimental effect to pulmonary PCM due to its suppressive effect on the innate and adaptive immunity resulting in progressive infection and precocious mortality of infected hosts.     

In Situ Patrolling of Regulatory T Cells Is Essential for Protecting Autoimmune Exocrinopathy

Background

Migration of T cells, including regulatory T (Treg) cells, into the secondary lymph organs is critically controlled by chemokines and adhesion molecules. However, the mechanisms by which Treg cells regulate organ-specific autoimmunity via these molecules remain unclear. Although we previously reported autoimmune exocrinopathy resembling Sjögren''s syndrome (SS) in the lacrimal and salivary glands from C-C chemokine receptor 7 (CCR7)-deficient mice, it is still unclear whether CCR7 signaling might specifically affect the dynamics and functions of Treg cells in vivo. We therefore investigated the cellular mechanism for suppressive function of Treg cells via CCR7 in autoimmunity using mouse models and human samples.

Methods and Findings

Patrolling Treg cells were detected in the exocrine organs such as lacrimal and salivary glands from normal mice that tend to be targets for autoimmunity while the Treg cells were almost undetectable in the exocrine glands of CCR7 ^−/− mice. In addition, we found the significantly increased retention of CD4⁺CD25⁺Foxp3⁺ Treg cells in the lymph nodes of CCR7 ^−/− mice with aging. Although Treg cell egress requires sphingosine 1-phosphate (S1P), chemotactic function to S1P of CCR7−/− Treg cells was impaired compared with that of WT Treg cells. Moreover, the in vivo suppression activity was remarkably diminished in CCR7 ^−/− Treg cells in the model where Treg cells were co-transferred with CCR7 ^−/− CD25^-CD4⁺ T cells into Rag2 ^−/− mice. Finally, confocal analysis showed that CCR7⁺Treg cells were detectable in normal salivary glands while the number of CCR7⁺Treg cells was extremely decreased in the tissues from patients with Sjögren''s syndrome.

Conclusions

These results indicate that CCR7 essentially governs the patrolling functions of Treg cells by controlling the traffic to the exocrine organs for protecting autoimmunity. Characterization of this cellular mechanism could have clinical implications by supporting development of new diagnosis or treatments for the organ-specific autoimmune diseases such as Sjögren''s syndrome and clarifying how the local immune system regulates autoimmunity.     

CXCR3-Dependent CD4+ T Cells Are Required to Activate Inflammatory Monocytes for Defense against Intestinal Infection

Chemokines and their receptors play a critical role in orchestrating immunity to microbial pathogens, including the orally acquired Th1-inducing protozoan parasite Toxoplasma gondii. Chemokine receptor CXCR3 is associated with Th1 responses, and here we use bicistronic CXCR3-eGFP knock-in reporter mice to demonstrate upregulation of this chemokine receptor on CD4⁺ and CD8⁺ T lymphocytes during Toxoplasma infection. We show a critical role for CXCR3 in resistance to the parasite in the intestinal mucosa. Absence of the receptor in Cxcr3^−/− mice resulted in selective loss of ability to control T. gondii specifically in the lamina propria compartment. CD4⁺ T cells were impaired both in their recruitment to the intestinal lamina propria and in their ability to secrete IFN-γ upon stimulation. Local recruitment of CD11b⁺Ly6C/G⁺ inflammatory monocytes, recently reported to be major anti-Toxoplasma effectors in the intestine, was not impacted by loss of CXCR3. However, inflammatory monocyte activation status, as measured by dual production of TNF-α and IL-12, was severely impaired in Cxcr3^−/− mice. Strikingly, adoptive transfer of wild-type but not Ifnγ^−/− CD4⁺ T lymphocytes into Cxcr3^−/− animals prior to infection corrected the defect in inflammatory macrophage activation, simultaneously reversing the susceptibility phenotype of the knockout animals. Our results establish a central role for CXCR3 in coordinating innate and adaptive immunity, ensuring generation of Th1 effectors and their trafficking to the frontline of infection to program microbial killing by inflammatory monocytes.     

Skewed Distribution of IL-7 Receptor-α-Expressing Effector Memory CD8+ T Cells with Distinct Functional Characteristics in Oral Squamous Cell Carcinoma

CD8⁺ T cells play important roles in anti-tumor immunity but distribution profile or functional characteristics of effector memory subsets during tumor progression are unclear. We found that, in oral squamous carcinoma patients, circulating CD8⁺ T cell pools skewed toward effector memory subsets with the distribution frequency of CCR7⁻CD45RA⁻CD8⁺ T cells and CCR7⁻ CD45RA⁺CD8⁺ T cells negatively correlated with each other. A significantly higher frequency of CD127^lo CCR7⁻CD45RA⁻CD8⁺ T cells or CCR7⁻CD45RA⁺CD8⁺ T cells among total CD8⁺ T cells was found in peripheral blood or tumor infiltrating lymphocytes, but not in regional lymph nodes. The CD127^hi CCR7⁻CD45RA⁻CD8⁺ T cells or CCR7⁻CD45RA⁺CD8⁺ T cells maintained significantly higher IFN-γ, IL-2 productivity and ex vivo proliferative capacity, while the CD127^lo CCR7⁻CD45RA⁻CD8⁺ T cells or CCR7⁻CD45RA⁺CD8⁺ T cells exhibited higher granzyme B productivity and susceptibility to activation induced cell death. A higher ratio of CCR7⁻CD45RA⁺CD8⁺ T cells to CCR7⁻CD45RA⁻CD8⁺ T cells was associated with advanced cancer staging and poor differentiation of tumor cells. Therefore, the CD127^lo CCR7⁻CD45RA⁻CD8⁺ T cells and CCR7⁻CD45RA⁺CD8⁺ T cells are functionally similar CD8⁺ T cell subsets which exhibit late differentiated effector phenotypes and the shift of peripheral CD8⁺ effector memory balance toward CCR7⁻CD45RA⁺CD8⁺ T cells is associated with OSCC progression.     

Chemokine Receptors CCR6 and CXCR3 Are Necessary for CD4+ T Cell Mediated Ocular Surface Disease in Experimental Dry Eye Disease

CD4⁺ T cells are essential to pathogenesis of ocular surface disease in dry eye. Two subtypes of CD4⁺ T cells, Th1 and Th17 cells, function concurrently in dry eye to mediate disease. This occurs in spite of the cross-regulation of IFN-γ and IL-17A, the prototypical cytokines Th1 and Th17 cells, respectively. Essential to an effective immune response are chemokines that direct and summon lymphocytes to specific tissues. T cell trafficking has been extensively studied in other models, but this is the first study to examine the role of chemokine receptors in ocular immune responses. Here, we demonstrate that the chemokine receptors, CCR6 and CXCR3, which are expressed on Th17 and Th1 cells, respectively, are required for the pathogenesis of dry eye disease, as CCR6KO and CXCR3KO mice do not develop disease under desiccating stress. CD4⁺ T cells from CCR6KO and CXCR3KO mice exposed to desiccating stress (DS) do not migrate to the ocular surface, but remain in the superficial cervical lymph nodes. In agreement with this, CD4⁺ T cells from CCR6 and CXCR3 deficient donors exposed to DS, when adoptively transferred to T cell deficient recipients manifest minimal signs of dry eye disease, including significantly less T cell infiltration, goblet cell loss, and expression of inflammatory cytokine and matrix metalloproteinase expression compared to wild-type donors. These findings highlight the important interaction of chemokine receptors on T cells and chemokine ligand expression on epithelial cells of the cornea and conjunctiva in dry eye pathogenesis and reveal potential new therapeutic targets for dry eye disease.     

Inducible Deletion of CD28 Prior to Secondary Nippostrongylus brasiliensis Infection Impairs Worm Expulsion and Recall of Protective Memory CD4+ T Cell Responses

IL-13 driven Th2 immunity is indispensable for host protection against infection with the gastrointestinal nematode Nippostronglus brasiliensis. Disruption of CD28 mediated costimulation impairs development of adequate Th2 immunity, showing an importance for CD28 during the initiation of an immune response against this pathogen. In this study, we used global CD28^−/− mice and a recently established mouse model that allows for inducible deletion of the cd28 gene by oral administration of tamoxifen (CD28^−/loxCre^+/−+TM) to resolve the controversy surrounding the requirement of CD28 costimulation for recall of protective memory responses against pathogenic infections. Following primary infection with N. brasiliensis, CD28^−/− mice had delayed expulsion of adult worms in the small intestine compared to wild-type C57BL/6 mice that cleared the infection by day 9 post-infection. Delayed expulsion was associated with reduced production of IL-13 and reduced serum levels of antigen specific IgG1 and total IgE. Interestingly, abrogation of CD28 costimulation in CD28^−/loxCre^+/− mice by oral administration of tamoxifen prior to secondary infection with N. brasiliensis resulted in impaired worm expulsion, similarly to infected CD28^−/− mice. This was associated with reduced production of the Th2 cytokines IL-13 and IL-4, diminished serum titres of antigen specific IgG1 and total IgE and a reduced CXCR5⁺ T_FH cell population. Furthermore, total number of CD4⁺ T cells and B220⁺ B cells secreting Th1 and Th2 cytokines were significantly reduced in CD28^−/− mice and tamoxifen treated CD28^−/loxCre^+/− mice compared to C57BL/6 mice. Importantly, interfering with CD28 costimulatory signalling before re-infection impaired the recruitment and/or expansion of central and effector memory CD4⁺ T cells and follicular B cells to the draining lymph node of tamoxifen treated CD28^−/loxCre^+/− mice. Therefore, it can be concluded that CD28 costimulation is essential for conferring host protection during secondary N. brasiliensis infection.     

Calcitonin Gene-Related Peptide Regulates Type IV Hypersensitivity through Dendritic Cell Functions

Dendritic cells (DCs) play essential roles in both innate and adaptive immune responses. In addition, mutual regulation of the nervous system and immune system is well studied. One of neuropeptides, calcitonin gene-related peptide (CGRP), is a potent regulator in immune responses; in particular, it has anti-inflammatory effects in innate immunity. For instance, a deficiency of the CGRP receptor component RAMP 1 (receptor activity-modifying protein 1) results in higher cytokine production in response to LPS (lipopolysaccharide). On the other hand, how CGRP affects DCs in adaptive immunity is largely unknown. In this study, we show that CGRP suppressed Th1 cell differentiation via inhibition of IL-12 production in DCs using an in vitro co-culture system and an in vivo ovalbumin-induced delayed-type hypersensitivity (DTH) model. CGRP also down-regulated the expressions of chemokine receptor CCR2 and its ligands CCL2 and CCL12 in DCs. Intriguingly, the frequency of migrating CCR2⁺ DCs in draining lymph nodes of RAMP1-deficient mice was higher after DTH immunization. Moreover, these CCR2⁺ DCs highly expressed IL-12 and CD80, resulting in more effective induction of Th1 differentiation compared with CCR2⁻ DCs. These results indicate that CGRP regulates Th1 type reactions by regulating expression of cytokines, chemokines, and chemokine receptors in DCs.