Source Of IFN-γ Research Articles

Dendritic cell-mediated responses to secreted Cryptosporidium effectors promote parasite-specific CD8+ T cell responses

Cryptosporidium causes debilitating diarrheal disease in patients with primary and acquired defects in T cell function. However, it has been a challenge to understand how this infection generates T cell responses and how they mediate parasite control. Here, Cryptosporidium was engineered to express a parasite effector protein (MEDLE-2) that contains the MHC-I restricted SIINFEKL epitope which is recognized by TCR transgenic OT-I CD8+ T cells. These modified parasites induced expansion of endogenous SIINFEKL-specific and OT-I CD8+ T cells that were a source of IFN-γ that could restrict growth of Cryptosporidium. This T cell response was dependent on the translocation of the effector and similar results were observed with another secreted parasite effector (ROP1). Although infection and these translocated effector proteins are restricted to intestinal epithelial cells (IEC), type I dendritic cells (cDC1) were required to generate CD8+ T cell responses to these model antigens. These data sets highlight Cryptosporidium effectors as potential targets of the immune system and suggest that crosstalk between enterocytes and cDC1s is crucial for CD8+ T cell responses to Cryptosporidium.

NK cells reduce anergic T cell development in early-stage tumors by promoting myeloid cell maturation.

Studies of NK cells in tumors have primarily focused on their direct actions towards tumor cells. We evaluated the impact of NK cells on expression of homing receptor ligands on tumor vasculature, intratumoral T cell number and function, and T cell activation in tumor draining lymph node. Using an implantable mouse model of melanoma, T cell responses and homing receptor ligand expression on the vasculature were evaluated with and without NK cells present during the early stages of the tumor response by flow cytometry. NK cells in early-stage tumors are one source of IFNγ that augments homing receptor ligand expression. More significantly, NK cell depletion resulted in increased numbers of intratumoral T cells with an anergic phenotype. Anergic T cell development in tumor draining lymph node was associated with increased T-cell receptor signaling but decreased proliferation and effector cell activity, and an incomplete maturation phenotype of antigen presenting cells. These effects of NK depletion were similar to those of blocking CD40L stimulation. We conclude that an important function of NK cells is to drive proper APC maturation via CD40L during responses to early-stage tumors, reducing development of anergic T cells. The reduced development of anergic T cells resulting in improved tumor control and T cell responses when NK cells were present.

Abstract 251: Inhibition of stromal p38MAPKalpha triggers innate-adaptive anti-tumor immunity in metastatic breast cancer

Abstract Metastases are the main cause of death in breast cancer patients. Among breast metastatic sites, bone is the most prevalent site that can also seed secondary metastases. Moreover, once tumor cells reach the bone, the disease is considered incurable and treatments are only palliative. For this reason, new therapies that limit metastatic growth are desperately needed. We previously showed that pharmacological inhibition of p38MAPKα (p38i) limited visceral and bone metastases in several clinically relevant models of breast cancer metastasis. Indeed, we found that this approach had no impact on tumor cells but instead limited the supporting nature of the stromal compartment. These findings led us to ask how p38i impacted the stromal compartment. Using a novel strategy to segregate tumor-infiltrating from non-tumor-infiltrating stromal cells in the bone metastatic site, we carried out single cell RNA sequencing (scRNA-Seq) to investigate how p38i altered immune and non-immune tumor stroma. Our analyses revealed that p38i treatment shifts tumor-associated macrophages in the bone towards a tumor-suppressive phenotype with increased signatures for IFNγ signaling and adaptive immunity. Importantly, the gene signature we identified in bone macrophages under p38i treatment correlated with better survival among patients with luminal B breast tumors. Given the importance of the p38i gene signature in patient samples, we next depleted macrophages and also blocked IFNγ and found both approaches reversed the p38i anti-tumor effect. Because T cells are an important source of IFNγ in vivo, we next assessed whether T cells were required by p38i to limit tumor growth. Depletion of CD4+ T cells, but not CD8+ T cells, blunted the p38i anti-tumor effect. Although p38i did not increase CD4+ T cell infiltration, p38i increased the percentage of activated CD4+ T cells infiltrating bone metastases. The requirement for CD4+ T cells in our model and the lack of evident OX40 ligand in our scRNA-Seq data led us to ask if p38i could be used in combination with immunotherapy to further limit metastases. Indeed, combination of p38i and agonist anti-OX40 (agOX-40) immunotherapy synergistically reduced metastatic growth and increased overall survival in our mouse models. Finally, because our work revealed no obvious role for CD8+ T cells in this model, we next asked if introduction of a CD8+ T cell antigen could further increase the anti-tumor effects of p38i. We thus modified PyMT-BO1 cells to express ovalbumin, which contains a strong CD8 antigen and combined it with p38i and agOX40. This combination led to a greater than 90% cure rate of mice with metastatic lesions. Although the mechanisms by which inhibition of p38MAPKα shapes the metastatic tumor microenvironment are still under investigation, our findings indicate that metastatic breast cancer patients may benefit from immunotherapy when carried out in combination with p38i. Citation Format: Douglas Vendas Faget, Xianmin Luo, Qihao Ren, Jiayu Ye, Thomas Cole, Zhangting Yao, Bhavna Murali, Xinming Su, Yalin Xu, Joseph B. Monahan, Katherine N. Weilbaecher, David G. DeNardo, Sheila A. Stewart. Inhibition of stromal p38MAPKalpha triggers innate-adaptive anti-tumor immunity in metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 251.

MO248P-GLYCOPROTEIN EXPRESSING IL-17A+IFN-GAMA+ TH17/1 CELLS ARE REFRACTORY TO GLUCOCORTICOID IN NEPHROTIC SYNDROME*

Abstract Background and Aims Th17 cells are critical effectors mediating the autoimmunity in nephrotic syndrome (NS). Elevated IFN-γ has also been involved in NS; however, it remains unclear to what extent Th1 cells contribute to glucocorticoids resistance in NS. P-glycoprotein (P-gp) effluxes glucocorticoids outside the cells and selectively expressed differentially on T cell subtypes. In this study, we investigated the role of P-gp and cellular source of IFN-γ and assessed its contribution to glucocorticoids resistance in NS. Method We analyzed the frequency of pathogenic IL-17A+IFN-γ+ Th17/1 lymphocytes and P-gp expression on their surface by flow cytometry in SSNS (n = 32; mean age: 9.06 ± 5.84) and SRNS (n = 28; mean age: 11.29 ± 3.73) patients. We also included 15 age- and sex-matched healthy controls. All patients were of biopsy proven minimal change disease and all patients were treated with steroids. All patients were recruited as per the criteria of ISKDC. Results We found a significant IL-17A+IFN-γ+ Th17/1 population (P < 0.001) in steroid resistant NS (SRNS) as compared to steroid sensitive NS (SSNS) patients. IL-12 and IL-23 are significantly higher in SRNS as compared to SSNS patients which are require for transition of pathogenic Th17 cells to IFN-γ producers. Of the IL-17A+IFN-γ+ Th17/1 population 95.8% cells were expressed P-gp on their surface in SRNS; however only 30.1% cells expressed P-gp in SSNS group (Figure 1). We also observed that P-gp expression correlate positively with IL-17A+IFN-γ+ Th17/1 population (r= 0.739, p< 0.001) significantly. Conclusion The above findings clearly show that higher expression of P-gp on IL-17A+IFN-γ+ Th17/1 cells associated with steroid resistance in nephrotic syndrome through both IL-17A and IFN-γ.

Invariant Natural Killer T Cells as Key Players in Host Resistance against Paracoccidioides brasiliensis.

Invariant Natural Killer T (iNKT) cells are key players in the immunity to several pathogens; however, their involvement in the resistance to Paracoccidioides brasiliensis infection remains unknown. Using splenocytes from CD1d (CD1d−/−) and iNKT-deficient (Jα18−/−) mice, we found that iNKT cells are the innate source of IFN-γ after P. brasiliensis infection and are required to potentiate macrophage oxidative burst and control fungal growth. To determine whether iNKT cells contribute in vivo to host resistance against P. brasiliensis infection, we infected intratracheally wild-type and Jα18−/− C57BL/6 mouse strains with the virulent Pb18 isolate. iNKT cell deficiency impaired the airway acute inflammatory response, resulting in decreased airway neutrophilia and reduced IFN-γ, KC, and nitric oxide (NO) production. The deficient innate immune response of Jα18−/− mice to Pb18 infection resulted in increased fungal burden in the lungs and spleen. Besides, the activation of iNKT cells in vivo by administration of the exogenous iNKT ligand α-galactosylceramide (α-GalCer) improved host resistance to P. brasiliensis infection. Although the mechanisms responsible for this phenomenon remain to be clarified, α-GalCer treatment boosted the local inflammatory response and reduced pulmonary fungal burden. In conclusion, our study is the first evidence that iNKT cells are important for the protective immunity to P. brasiliensis infection and their activation by an exogenous ligand is sufficient to improve the host resistance to this fungal infection.

Combined neutralization of interferon gamma and tumor necrosis factor alpha induces IL-4 production but has no direct additive impact on parasite burden in splenic cultures of human visceral leishmaniasis.

Immune activating cytokines Interferon (IFN)-γ and Tumor necrosis factor (TNF)-α are known to activate macrophages for killing of Leishmania parasite. IFN-γ provides therapeutic potential while TNF-α has been recognized to mediate protection in visceral model of infection. In the present study we investigated whether combination of IFN-γ and TNF-α has better therapeutic strength than individually using one of these cytokines in Visceral Leishmaniasis (VL) patients. We performed combined blockade of IFN-γ and TNF-α in VL splenic biopsies and demonstrated it’s impact on number of viable amastigotes and cytokine production. Additionally, selective depletion of splenic cell subsets was performed to establish the cellular sources of IFN-γ and TNF-α. Treatment of splenic aspirate cells with combination of anti-IFN-γ and anti-TNF-α monoclonal antibodies for 72 hours enabled no direct additive impact of these cytokines on parasite replication and IL-10 secretion, but IL-4 production was induced. Further assessment of splenic biopsies put forward CD4+ T cells as a source of IFN-γ whereas CD14+ cells contribute towards TNF-α production. Overall our results suggest, the interplay of pro-inflammatory cytokines IFN-γ derived from CD4+T lymphocytes and TNF-α from CD14+ cells has no direct additive impact on parasite replication but induces IL-4 production. Our data does not support direct targeting of IFN-γ and TNF-α for combination therapy but targeting these cytokines as an adjuvant in patients with exaggerated tissue inflammatory responses can have favourable patient outcome.

Quantification of NK cell activity using whole blood: Methodological aspects of a new test

Natural Killer (NK) cells are essential in the biological fight against cancer and intracellular pathogens, and their level of activity has in many settings been used as a biomarker for a functional immune response. Currently, NK cell activity is measured using either 51Cr-release assays or flow cytometry based assays revealing the cells´ cytotoxic capacity or by stimulating them to produce cytokines. Although very effective, these are cumbersome techniques not suitable for high volume clinical laboratories.Recently, an assay has been introduced to measure NK cell activity in a simple and standardized manner. Following stimulation of NK cells in whole blood with a recombinant protein, it utilizes the concentration of IFNγ released to the plasma as a surrogate marker for NK cell activity. However, whole blood holds several sources of IFNγ which may blur the results and hamper the interpretation of the test. Therefore, the present study aimed at analyzing how specifically the test is measuring the activity of NK cells.Intracellular flow cytometry showed that NK cells, T cells, and Natural Killer T (NKT) cells were producing IFNγ in the assay, however when analyzing the distribution of lymphocytes in the IFNγ-expressing subset, the proportion of NK cells far exceeded the percentage of T-, and NKT cells (p < .0001). Hence, our data indicate that the readout of the test was indicative of the NK cells´ ability to mount a response and thus the results may pave the way for the assay to become applicable in the clinical setting as an estimate of NK cell activity for both diagnostic and prognostic purposes.

Evolution of T Cell Responses during Measles Virus Infection and RNA Clearance

Measles is an acute viral disease associated both with immune suppression and development of life-long immunity. Clearance of measles virus (MeV) involves rapid elimination of infectious virus during the rash followed by slow elimination of viral RNA. To characterize cellular immune responses during recovery, we analyzed the appearance, specificity and function of MeV-specific T cells for 6 months after respiratory infection of rhesus macaques with wild type MeV. IFN-γ and IL-17-producing cells specific for the hemagglutinin and nucleocapsid proteins appeared in circulation in multiple waves approximately 2-3, 8 and 18–24 weeks after infection. IFN-γ-secreting cells were most abundant early and IL-17-secreting cells late. Both CD4+ and CD8+ T cells were sources of IFN-γ and IL-17, and IL-17-producing cells expressed RORγt. Therefore, the cellular immune response evolves during MeV clearance to produce functionally distinct subsets of MeV-specific CD4+ and CD8+ T cells at different times after infection.

Neutrophils and Ly6Chi monocytes collaborate in generating an optimal cytokine response that protects against pulmonary Legionella pneumophila infection.

Early responses mounted by both tissue-resident and recruited innate immune cells are essential for host defense against bacterial pathogens. In particular, both neutrophils and Ly6Chi monocytes are rapidly recruited to sites of infection. While neutrophils and monocytes produce bactericidal molecules, such as reactive nitrogen and oxygen species, both cell types are also capable of synthesizing overlapping sets of cytokines important for host defense. Whether neutrophils and monocytes perform redundant or non-redundant functions in the generation of anti-microbial cytokine responses remains elusive. Here, we sought to define the contributions of neutrophils and Ly6Chi monocytes to cytokine production and host defense during pulmonary infection with Legionella pneumophila, responsible for the severe pneumonia Legionnaires’ disease. We found that both neutrophils and monocytes are critical for host defense against L. pneumophila. Both monocytes and neutrophils contribute to maximal IL-12 and IFNγ responses, and monocytes are also required for TNF production. Moreover, natural killer (NK) cells, NKT cells, and γδ T cells are sources of IFNγ, and monocytes direct IFNγ production by these cell types. Thus, neutrophils and monocytes cooperate in eliciting an optimal cytokine response that promotes effective control of bacterial infection.

T-bet-Expressing B Cells Are Positively Associated with Crohn's Disease Activity and Support Th1 Inflammation.

Pathogenesis of Crohn's disease (CD) is thought to involve the chronic activation of T helper 1 (Th1)- and Th17-mediated inflammation, such as the production of interferon-gamma (IFN-γ) and interleukin 17 (IL-17). However, studies have also shown that although IFN-γ is required, IFN-γ-producing or T-bet-expressing Th1 cells are dispensable. We therefore examined T-bet-expressing B cells as another source of IFN-γ that potentially supported intestinal inflammation in CD patients. We found that the frequencies of T-bet-expressing B cells were significantly upregulated and abundantly present in the gut of active, but not quiescent, CD patients. The frequencies of T-bet-expressing B cells were also directly correlated with CD disease activity. These T-bet+ B cells were almost exclusively IgG expressing and produced significantly higher amounts of IFN-γ, IL-6, and IL-12 than IgA- and IgM-expressing T-bet- B cells. These B cells also supported IFN-γ production of CD4+ T cells. T-bet expression was induced in vitro in peripheral blood B cells through the stimulation of B-cell receptor (BCR), Toll-like receptor 7 (TLR7), and IFN-γ, which resembled gut T-bet+ B cells in terms of elevated IFN-γ. We found that these stimulated B cells, but not unstimulated B cells, supported the IFN-γ and IL-12 production from autologous CD4+ T cells. In addition, in patients with higher gut T-bet+ B-cell percentage, a higher frequency of gut-infiltrating IFN-γ+ and IL-12+ T cells was also observed. Together, our results suggested that T-bet-expressing B cells could contribute to the intestinal Th1 inflammation in CD patients.

Different response to glucocorticoid therapy in autoimmune diseases of CNS

Th17 cells and interleukin (IL-17), their signature cytokine, have the main role in the pathogenesis of autoimmune diseases of the central nervous system such as multiple sclerosis and experimental autoimmune encephalomyelitis (EAE). The effect of glucocorticoids (GC) on expression and production of IL-17 has not been thoroughly tested yet. Also, the site of action of GC is not precisely defined. This paper presents the main results of the Doctoral thesis devoted to studies of GC on the production of IL-17 in the model of EAE, induced in susceptible laboratory animals. Methylprednisolone (MP), a synthetic glucocorticoid, inhibit in vitro production of IL-17 in mitogen-stimulated lymph node cells (LNC) as well as in myelin basic protein (MBP)-stimulated draining LNC in dose- dependent manner. However, under the same conditions inhibitory effect of the MP on production and expression of the genes for IFN-γ, a cytokine that TH1 cells generate, is significantly more pronounced. Interestingly, when we analyzed effects of MP applied in vivo in EAE, the same phenomenon was observed: the proportion of IFN-γ producing, but not all of IL-17 cells were reduced in cells isolated from MP treated rats in comparison to control rats which indicates that MP achieves its effects not only in the peripheral lymphoid tissues, but also in target tissue. Different sensitivities of Th1 and Th17 cells that are major cellular sources of IFN-γ or IL-17 in the effect of the GC has been observed in other animal models and in human disease. Understanding the molecular mechanisms underlying the relative resistance of Th17 cells on the operation of GC is very important for the development of new strategies in the treatment of those forms of autoimmune and chronic diseases that are resistant to the effect of glucocorticoids.

A novel experimental model of Cryptococcus neoformans‐related immune reconstitution inflammatory syndrome (IRIS) provides insights into pathogenesis

Antiretroviral therapy (ART) has yielded major advances in fighting the HIV pandemic by restoring protective immunity. However, a significant proportion of HIV patients co-infected with the opportunistic fungal pathogen Cryptococcus neoformans paradoxically develops a life-threatening immune reconstitution inflammatory syndrome (IRIS) during antiretroviral therapy. Despite several clinical studies, the underlying pathomecha-nisms are poorly understood. Here, we present the first mouse model of cryptococcal IRIS that allows for a detailed analysis of disease development. Lymphocyte-deficient RAG-1(-/-) mice are infected with C. neoformans and 4 weeks later adoptively transferred with purified CD4(+) T cells. Reconstitution of CD4(+) T cells is sufficient to induce a severe inflammatory disease similar to clinical IRIS in C. neoformans-infected RAG-1(-/-) mice of different genetic backgrounds and immunological phenotypes (i.e. C57BL/6 and BALB/c). Multiorgan inflammation is accompanied by a systemic release of distinct proinflammatory cytokines, i.e. IFN-γ, IL-6, and TNF-α. IRIS development is characterized by infection-dependent activation of donor CD4(+) T cells, which are the source of IFN-γ. Interestingly, IFN-γ-mediated effects are not required for disease induction. Taken together, this novel mouse model of cryptococcal IRIS provides a useful tool to verify potential mechanisms of pathogenesis, revealing targets for diagnosis and therapeutic interventions.

Type I, II, and III Interferons: Regulating Immunity to Mycobacterium tuberculosis Infection.

Interferons (IFNs) are cytokines released by host cells in response to the presence of pathogens or tumor cells. The aim of this review was to present the previously known and new findings about the role of interferons type I and II, and recently discovered type III in Mycobacterium tuberculosis (M. tuberculosis) infection control. Infection of various cell types with M. tuberculosis induce both IFN-α and IFN-β synthesis. The majority of the studies support the findings that IFN type I actually promotes infection with M. tuberculosis. It has been well establish that IFN-γ has protective function against M. tuberculosis and the other mycobacteria and that the primary source of this cytokine are CD4(+) and CD8(+) T cells. Recently, it has been shown that also the innate lymphocytes, γδ T cells, natural killer (NK) T cells, and NK cells can also be the source of IFN-γ in response to mycobacterial infection. Several studies have shown that CD4(+) T cells protect mice against M. tuberculosis independently of IFN-γ. The balance between IFN-γ and different cytokines such as IL-10 and other Th2 cell cytokines is likely to influence disease outcome. Type I IFN appears to be detrimental through at least three separate, but overlapping, type I IFN-mediated mechanisms: induction of excessive apoptosis, specific suppression of Th1 and IFN-γ responses, and dampening of the immune response by strong IL-10 induction. Recently it has been found that M. tuberculosis infection in A549 lung epithelial cells stimulate up-regulation of IFN-λ genes in vitro. IFN-λs also have a role in modulation of Th1/Th2 response. IFN-λs are not essential for M. tuberculosis infection control, but can give some contribution in immune response to this pathogen.

Abstract 051: The Role of Immunological Memory in Hypertension

Immunological memory provides protection to repeated antigen challenges and is a cardinal feature of adaptive immunity. We have previously shown that adaptive immunity contributes to hypertension and have observed memory T cells in several models. We hypothesized that memory T cells contribute to long-term renal damage in response to repeated hypertensive challenges. To impose repeated episodes of hypertension, we treated C57BL/6 mice with L-NAME (0.5mg/ml) in drinking water for two weeks, allowed a two-week normotensive interval and then fed high salt (4% NaCl) for three weeks. L-NAME followed by high salt increased SBP to 151 ± 14 mmHg and caused a two-fold increase in CD4 + and CD8 + memory T cells in the kidney and bone marrow, as identified by the surface marker CD44hi. Intracellular staining showed that memory T cells were predominant sources of the inflammatory cytokines IL-17A and IFN-γ. Development and reactivation of memory T cells require the interaction of CD27 on T cells with CD70 on antigen presenting cells. Flow cytometry revealed that L-NAME/High salt increased expression of CD70 on splenic macrophages by 5-fold and dendritic cells by 3-fold. Because memory T cells are a major source of IFN-γ, we examined the hypertensive response to the L-NAME/high salt protocol in IFN-γ -/- mice. The hypertension caused by L-NAME was identical between WT, CD70 -/- and IFN-γ -/- mice. In contrast, the hypertension induced by subsequent salt administration was markedly attenuated in CD70 -/- mice (123 ± 1.3 mmHg, p&lt;0.02). Likewise, mice lacking IFN-γ developed blunted hypertension during the salt-feeding phase (127.6 ± 5.5 mmHg, p&lt;0.04). Interestingly, CD70 -/- and IFN-γ -/- mice failed to develop memory T cell formation in the kidney. The L-NAME/high salt caused striking albuminuria and increased urinary N-gal in WT mice, and these were absent in CD70 -/- and IFN-γ -/- mice. In contrast, L-NAME/high salt had no effect on renal angiotensinogen levels. Thus, repeated hypertensive stimuli lead to accumulation of long-lived effector memory T cells that are major sources of inflammatory cytokines, which in turn promote renal dysfunction, salt sensitivity and hypertension. These studies provide further insight into how the adaptive immune system promotes hypertension.

Mechanisms of Interferon-γ Production by Neutrophils and Its Function duringStreptococcus pneumoniaePneumonia

Bacterial pneumonia is a common public health problem associated with significant mortality, morbidity, and cost. Neutrophils are usually the earliest leukocytes to respond to bacteria in the lungs. Neutrophils rapidly sequester in the pulmonary microvasculature and migrate into the lung parenchyma and alveolar spaces, where they perform numerous effector functions for host defense. Previous studies showed that migrated neutrophils produce IFN-γ early during pneumonia induced by Streptococcus pneumoniae and that early production of IFN-γ regulates bacterial clearance. IFN-γ production by neutrophils requires Rac2, Hck/Lyn/Fgr Src family tyrosine kinases, and NADPH oxidase. Our current studies examined the mechanisms that regulate IFN-γ production by lung neutrophils during acute S. pneumoniae pneumonia in mice and its function. We demonstrate that IFN-γ production by neutrophils is a tightly regulated process that does not require IL-12. The adaptor molecule MyD88 is critical for IFN-γ production by neutrophils. The guanine nucleotide exchange factor CalDAG-GEFI modulates IFN-γ production. The CD11/CD18 complex, CD44, Toll-like receptors 2 and 4, TRIF, and Nrf2 are not required for IFN-γ production by neutrophils. The recently described neutrophil-dendritic cell hybrid cell, identified by its expression of Ly6G and CD11c, is present at low numbers in pneumonic lungs and is not a source of IFN-γ. IFN-γ produced by neutrophils early during acute S. pneumoniae pneumonia induces transcription of target genes in the lungs, which are critical for host defense. These studies underline the complexity of the neutrophil responses during pneumonia in the acute inflammatory response and in subsequent resolution or initiation of immune responses.

Abstract 22: Interferon-γ produced by tumor-infiltrating NK cells and CD4+ T cells downregulates TNFSF15 expression in endothelial cells

Abstract Endothelial cells in an established vasculature secrete tumor necrosis factor superfamily-15 (TNFSF15; VEGI; TL1A) that functions as a negative modulator of neovascularization to maintain blood vessel stability. TNFSF15 gene expression diminishes at angiogenesis and inflammation sites such as in cancers and wounds. Interferon-γ (IFNγ) is a pleiotropic cytokine expressed primarily by lymphocytes of T cell and NK cell lineages. IFNγ may mediate anti-tumor effects either directly on cancer cell growth or indirectly through immunomodulatory and antiangiogenic responses. However, it was also reported that IFNγ promotes angiogenesis under certain circumstances. Here we show that IFNγ suppresses TNFSF15 expression in human umbilical cord vein endothelial cells (HUVEC). This activity is mediated by IFNγ receptor and the transcription factor STAT1. Since tumor-infiltrating NK and CD4+ T cells are the main sources of IFNγ in tumor lesions, we isolated these cells from peripheral blood of healthy individuals, treated the cells with ovarian cancer OVCAR3 cell-conditioned media, and found a 1- and 10-fold increase of IFNγ production in NK and CD4+ T cells, respectively, compared with that in vehicle-treated cells. These findings support the view that tumor-infiltrating NK and CD4+ T cells under the influence of cancer cells significantly increase the production of IFNγ, which in turn inhibits TNFSF15 expression in tumor vascular endothelial cells, shifting the balance of pro- and anti-angiogenic factors toward escalated angiogenesis potential in the tumor. Citation Format: Xin Gu, Tao Cheng, Luyuan Li. Interferon-γ produced by tumor-infiltrating NK cells and CD4+ T cells downregulates TNFSF15 expression in endothelial cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 22. doi:10.1158/1538-7445.AM2014-22

Induction of interferon-gamma and downstream pathways during establishment of fetal persistent infection with bovine viral diarrhea virus

Development of transplacental infection depends on the ability of the virus to cross the placenta and replicate within the fetus while counteracting maternal and fetal immune responses. Unfortunately, little is known about this complex process. Non-cytopathic (ncp) strains of bovine viral diarrhea virus (BVDV), a pestivirus in the Flaviviridae family, cause persistent infection in early gestational fetuses (<150 days; persistently infected, PI), but are cleared by immunocompetent animals and late gestational fetuses (>150 days; transiently infected, TI). Evasion of innate immune response and development of immunotolerance to ncp BVDV have been suggested as possible mechanisms for the establishment of the persistent infection. Previously we have observed a robust temporal induction of interferon (IFN) type I (innate immune response) and upregulation of IFN stimulated genes (ISGs) in BVDV TI fetuses. Modest chronic upregulation of ISGs in PI fetuses and calves reflects a stimulated innate immune response during persistent BVDV infection. We hypothesized that establishing persistent fetal BVDV infection is also accompanied by the induction of IFN-gamma (IFN-γ). The aims of the present study were to determine IFN-γ concentration in blood and amniotic fluid from control, TI and PI fetuses during BVDV infection and analyze induction of the IFN-γ downstream pathways in fetal lymphoid tissues. Two experiments with in vivo BVDV infections were completed. In Experiment 1, pregnant heifers were infected with ncp BVDV type 2 on day 75 or 175 of gestation or kept naïve to generate PI, TI and control fetuses, respectively. Fetuses were collected by Cesarean section on day 190. In Experiment 2, fetuses were collected on days 82, 89, 97, 192 and 245 following infection of pregnant heifers on day 75 of gestation. The results were consistent with the hypothesis that ncp BVDV infection induces IFN-γ secretion during acute infection in both TI and PI fetuses and that lymphoid tissues such as spleen, liver and thymus, serve both as possible sources of IFN-γ and target organs for its effects. Notably, induction of IFN-γ coincides with a decrease in BVDV RNA concentrations in PI fetal blood and tissues. This is the first report indicating the possible presence of an adaptive immune response in persistent BVDV infections, which may be contributing to the observed reduction of viremia in PI fetuses.

The SaeR/S two-component system induces interferon-gamma production in neutrophils during invasive Staphylococcus aureus infection

Invasive Staphylococcus aureus (S. aureus) disease is associated with neutrophil activity and pro-inflammatory cytokine expression, including interferon-gamma (IFNγ). Using a mouse model of S. aureus peritonitis, we identify neutrophils as the predominant source of IFNγ and link this induction with the SaeR/S two-component gene regulatory system. Relative to wild-type (BALB/c) mice, IFNγ-deficient mice demonstrated increased bacterial clearance and reduced cellular cytotoxicity following intraperitoneal challenge with S. aureus. Interestingly, bacterial burden and cytotoxicity were similar in BALB/c and IFNγ-deficient mice when infected with an isogenic saeR/S mutant strain. These findings suggest saeR/S-mediated neutrophil-derived IFNγ diminishes innate antibacterial mechanisms against S. aureus.

The IL-10-/- mouse as a model of chronic Lyme arthritis. (169.16)

Abstract IL-10 suppresses the development of arthritis in Borrelia burgdorferi infected mice. Infected IL-10-/- mice were previously found to have a robust and sustained interferon-inducible response in joint tissue, and elevated IFN-γ in sera. Treatment of IL-10-/- mice with anti-IFN-γ reduced arthritis severity and suppressed interferon-inducible transcripts to wild type levels, thereby linking dysregulation of IFN-γ to disease in the IL-10-/- mouse. Arthritis in IL-10 deficient mice was associated with elevated numbers of NK cell, NKT cell, α/β T cell, and macrophage infiltration of the infected joint. FACS lineage sorting revealed NK cells and CD4+ T cells as sources of IFN-γ in the joint tissue of IL-10-/- mice. These findings suggest the presence of a positive feedback loop in the joint tissue of infected IL-10-/- mice, where production of inflammatory chemokines, infiltration of IFN-γ producing cells, and additional production of inflammatory cytokines result in arthritis. This mechanism of arthritis is in contrast to that seen in C3H mice, where arthritis development is linked to transient production of Type I interferon, and develops independently of IFN-γ. Due to the sustained interferon response driven by NK cells and T cells, we propose the IL-10-/- mouse as a potential model to study the prolonged symptoms observed in some human Lyme disease patients.

Localized Production of IL-10 Suppresses Early Inflammatory Cell Infiltration and Subsequent Development of IFN-γ–Mediated Lyme Arthritis

IL-10 is a nonredundant inflammatory modulator that suppresses arthritis development in Borrelia burgdorferi-infected mice. Infected C57BL/6 (B6) IL-10(-/-) mice were previously found to have a prolonged IFN-inducible response in joint tissue. Infection of B6 IL-10 reporter mice identified macrophages and CD4(+) T cells as the primary sources of IL-10 in the infected joint tissue, suggesting that early local production of IL-10 dampened the proarthritic IFN response. Treatment of B6 IL-10(-/-) mice with anti-IFN-γ reduced the increase in arthritis severity and suppressed IFN-inducible transcripts to wild-type levels, thereby linking dysregulation of IFN-γ to disease in the B6 IL-10(-/-) mouse. Arthritis in B6 IL-10(-/-) mice was associated with elevated numbers of NK cell, NKT cell, α/β T cell, and macrophage infiltration of the infected joint. FACS lineage sorting revealed NK cells and CD4(+) T cells as sources of IFN-γ in the joint tissue of B6 IL-10(-/-) mice. These findings suggest the presence of a positive-feedback loop in the joint tissue of infected B6 IL-10(-/-) mice, in which production of inflammatory chemokines, infiltration of IFN-γ-producing cells, and additional production of inflammatory cytokines result in arthritis. This mechanism of arthritis is in contrast to that seen in C3H/He mice, in which arthritis development is linked to transient production of type I IFN and develops independently of IFN-γ. Due to the sustained IFN response driven by NK cells and T cells, we propose the B6 IL-10(-/-) mouse as a potential model to study the persistent arthritis observed in some human Lyme disease patients.