IL‐33 promotes innate IFN‐γ production and modulates dendritic cell response in LCMV‐induced hepatitis in mice

Recent studies have revealed IL‐33 as a key factor in promoting antiviral T‐cell responses. However, it is less clear as to how IL‐33 regulates innate immunity. In this study, we infected wild‐type (WT) and IL‐33^?/? mice with lymphocytic choriomeningitis virus and demonstrated an essential role of infection‐induced IL‐33 expression for robust innate IFN‐γ production in the liver. We first show that IL‐33 deficiency resulted in a marked reduction in the number of IFN‐γ⁺ γδ T and NK cells, but an increase in that of IL‐17⁺ γδ T cells at 16 h postinfection. Recombinant IL‐33 (rIL‐33) treatment could reverse such deficiency via increasing IFN‐γ‐producing γδ T and NK cells, and inhibiting IL‐17⁺ γδ T cells. We also found that rIL‐33‐induced type 2 innate lymphoid cells were not involved in T‐cell responses and liver injury, since the adoptive transfer of type 2 innate lymphoid cells neither affected the IFN‐γ and TNF‐α production in T cells, nor liver transferase levels in lymphocytic choriomeningitis virus infected mice. Interestingly, we found that while IL‐33 was not required for costimulatory molecule expression, it was critical for DC proliferation and cytokine production. Together, this study highlights an essential role of IL‐33 in regulating innate IFN‐γ‐production and DC function during viral hepatitis.     

IL‐18‐induced expression of high‐affinity IL‐2R on murine NK cells is essential for NK‐cell IFN‐γ production during murine Plasmodium yoelii infection

Early production of pro‐inflammatory cytokines, including IFN‐γ, is essential for control of blood‐stage malaria infections. We have shown that IFN‐γ production can be induced among human natural killer (NK) cells by coculture with Plasmodium falciparum infected erythrocytes, but the importance of this response is unclear. To further explore the role of NK cells during malaria infection, we have characterized the NK‐cell response of C57BL/6 mice during lethal (PyYM) or nonlethal (Py17XNL) P. yoelii infection. Ex vivo flow cytometry revealed that NK cells are activated within 24 h of Py17XNL blood‐stage infection, expressing CD25 and producing IFN‐γ; this response was blunted and delayed during PyYM infection. CD25 expression and IFN‐γ production were highly correlated, suggesting a causal relationship between the two responses. Subsequent in vitro experiments revealed that IL‐18 signaling is essential for induction of CD25 and synergizes with IL‐12 to enhance CD25 expression on splenic NK cells. In accordance with this, Py17XNL‐infected erythrocytes induced NK‐cell CD25 expression and IFN‐γ production in a manner that is completely IL‐18‐ and partially IL‐12‐dependent, and IFN‐γ production is enhanced by IL‐2. These data suggest that IL‐2 signaling via CD25 amplifies IL‐18‐ and IL‐12‐mediated NK‐cell activation during malaria infection.     

Activated natural killer cells accelerate liver damage in patients with chronic hepatitis B virus infection

Emerging evidence indicates that natural killer (NK) cells may contribute to liver injury in patients with hepatitis B virus (HBV) infection. Because HBV infection progresses through various disease phases, the cytolytic profiles of peripheral and intrahepatic NK cells in HBV‐infected patients remain to be defined. In this study, we comprehensively characterized intrahepatic and peripheral NK cells in a cohort of HBV‐infected individuals, and investigated their impact on liver pathogenesis during chronic HBV infection. The study population included 34 immune‐clearance (IC) patients, 36 immune‐tolerant (IT) carriers and 10 healthy subjects. We found that the activity of peripheral NK cells from IC patients was functionally elevated compared to IT carriers and controls, and NK cell activation was indicated by an increased expression of CD69, CD107a, interferon (IFN)‐γ and tumour necrosis factor (TNF)‐α. Further analysis showed that the increased activity of both peripheral and hepatic NK cells was correlated positively with liver injury, which was assessed by serum alanine aminotransferase levels (ALT) and the liver histological activity index (HAI). Interestingly, the frequency of peripheral NK cells was reduced in IC patients (especially those with higher HAI scores of 3–4), but there was a concomitant increase in hepatic NK cells. The functionally activated NK cells are enriched preferentially in the livers of IC patients and skew towards cytolytic activity that accelerates liver injury in chronic hepatitis B (CHB) patients.     

Siglec‐1 inhibits RSV‐induced interferon gamma production by adult T cells in contrast to newborn T cells

Interferon gamma (IFN‐γ) plays an important role in the antiviral immune response during respiratory syncytial virus (RSV) infections. Monocytes and T cells are recruited to the site of RSV infection, but it is unclear whether cell‐cell interactions between monocytes and T cells regulate IFN‐γ production. In this study, micro‐array data identified the upregulation of sialic acid‐binding immunoglobulin‐type lectin 1 (Siglec‐1) in human RSV‐infected infants. In vitro, RSV increased expression of Siglec‐1 on healthy newborn and adult monocytes. RSV‐induced Siglec‐1 on monocytes inhibited IFN‐γ production by adult CD4⁺ T cells. In contrast, IFN‐γ production by RSV in newborns was not affected by Siglec‐1. The ligand for Siglec‐1, CD43, is highly expressed on adult CD4⁺ T cells compared to newborns. Our data show that Siglec‐1 reduces IFN‐γ release by adult T cells possibly by binding to the highly expressed CD43. The Siglec‐1‐dependent inhibition of IFN‐γ in adults and the low expression of CD43 on newborn T cells provides a better understanding of the immune response against RSV in early life and adulthood.     

Enhancement of cytokine‐driven NK cell IFN‐γ production after vaccination of HCMV infected Africans

Human cytomegalovirus (HCMV) infection drives the phenotypic and functional differentiation of NK cells, thereby influencing the responses of these cells after vaccination. NK cell functional differentiation is particularly advanced in African populations with universal exposure to HCMV. To investigate the impact of advanced differentiation on vaccine‐induced responses, we studied NK‐cell function before and after vaccination with Trivalent Influenza Vaccine (TIV) or diphtheria, tetanus, pertussis, inactivated poliovirus vaccine (DTPiP) in Africans with universal, lifelong HCMV exposure. In contrast to populations with lower prevalence of HCMV infection, no significant enhancement of NK‐cell responses (IFN‐γ, CD107a, CD25) occurred after in vitro re‐stimulation of post‐vaccination NK cells with TIV or DTPiP antigens compared to pre‐vaccination baseline cells. However, both vaccinations resulted in higher frequencies of NK cells producing IFN‐γ in response to exogenous IL‐12 with IL‐18, which persisted for up to 6 months. Enhanced cytokine responsiveness was restricted to less differentiated NK cells, with increased frequencies of IFN‐γ⁺ cells observed within CD56^brightCD57^?, CD56^dimCD57^?NKG2C^? and CD56^dimCD57^?NKG2C⁺ NK‐cell subsets. These data suggest a common mechanism whereby different vaccines enhance NK cell IFN‐γ function in HCMV infected donors and raise the potential for further exploitation of NK cell “pre‐activation” to improve vaccine effectiveness.     

Innate IFN‐γ Production by Subsets of Natural Killer Cells,Natural Killer T Cells and γδ T Cells in Response to Dying Bacterial‐Infected Macrophages

Interferon‐γ (IFN‐γ) activation of macrophages is a crucial step in the early innate defence against bacterial infection. This innate IFN‐γ is thought to be produced mainly by natural killer (NK) cells through activation with interleukin (IL)‐12p70 secreted by macrophages and dendritic cells (DCs) that have sensed bacterial products. However, a number of reports have shown that bacterial stimuli are unable to induce macrophages and/or DCs to produce sufficient amounts of IL‐12p70 unless these cells are primed by IFN‐γ. It remains, therefore, unsettled how initial IFN‐γ is produced. In a previous study, we reported a novel IFN‐γ production pathway that was associated with cell death in macrophages caused by intracellular bacteria like Listeria monocytogenes (LM) and Shigella flexneri. In this study, we showed that cell death of bone‐marrow‐derived macrophage (BMM) cells following in vitro infection with Staphylococcus aureus (SA), an extracellular bacterium, can also stimulate this IFN‐γ production pathway. We also unequivocally demonstrated by using BMM cells from IL‐12‐deficient mice that the bacterial‐infected macrophage cell death‐mediated IFN‐γ production can occur without IL‐12 although the magnitude of the response is much smaller than that in the presence of IL‐12. The enhancing effect of IL‐12 on this response proved to be attributable to the negligible amounts (0.5～1.5 pg/ml) of IL‐12p70 but not to the large amounts of IL‐12p40 that were both secreted by SA‐ and LM‐infected macrophages. Taken all together, we propose that macrophage cell death caused by bacteria may trigger the initial IFN‐γ production at an early stage of bacterial infection.     

Activating NK cell receptor expression/function (NKp30, NKp46, DNAM-1) during chronic viraemic HCV infection is associated with the outcome of combined treatment

Specific NK cell killer inhibitory receptor (KIR):HLA haplotype combinations have been associated with successful clearance of acute and chronic HCV infection. Whether an imbalance of activating NK cell receptors also contributes to the outcome of treatment of chronic HCV infection, however, is not known. We studied peripheral NK cell phenotype and function in 28 chronically viraemic HCV genotype I treatment‐naïve patients who underwent treatment with pegylated IFN‐α and ribavirin. At baseline, chronically infected patients with sustained virological response (SVR) had reduced CD56^brightCD16^+/? cell populations, increased CD56^dullCD16⁺ NK cell proportions, and lower expression of NKp30, DNAM‐1, and CD85j. Similarly, reduced NK cell IFN‐γ production but increased degranulation was observed among nonresponding (NR) patients. After treatment, CD56^brightCD16^+/? NK cell numbers increased in both SVR and NR patients, with a parallel significant increase in activating NKp30 molecule densities in SVR patients only. In vitro experiments using purified NK cells in the presence of rIL‐2 and IFN‐α confirmed upregulation of NKp30 and also of NKp46 and DNAM‐1 in patients with subsequent SVR. Thus, differences in patient NK cell receptor expression and modulation during chronic HCV‐1 infection are associated with subsequent outcome of standard treatment. Individual activating receptor expression/function integrates with KIR:HLA genotype carriage to determine the clearance of HCV infection upon treatment.     

NK cells generate memory‐type responses to human cytomegalovirus‐infected fibroblasts

Natural killer (NK) cells are cytotoxic lymphocytes that selectively respond against abnormal cells. Human cytomegalovirus (HCMV) infection causes expansion of NKG2C⁺CD57⁺ NK cells in vivo and NKG2C⁺ NK cells proliferate when cultured with HCMV‐infected cells. This raises the possibility of an NK‐cell subset selectively responding against a specific pathogen and accruing memory. To test this possibility, we compared proliferation, natural cytotoxicity and interferon‐γ (IFN‐γ) production of NK cells from HCMV‐seropositive and HCMV‐seronegative individuals co‐cultured with HCMV‐infected or uninfected MRC‐5 cells. There was no significant difference in proliferation of NK cells from HCMV‐seropositive or seronegative individuals against uninfected MRC‐5 cells, but significantly more NK cells from the HCMV‐seropositive group proliferated in response to HCMV‐infected MRC‐5 cells. Natural cytotoxicity of NK cells against K562 cells increased following co‐culture with HCMV‐infected versus uninfected MRC‐5 only for the HCMV‐seropositive group. After co‐culture with HCMV‐infected MRC‐5 cells, proliferating NK cells from HCMV‐seropositive donors selectively produced IFN‐γ when re‐exposed to HCMV‐infected MRC‐5 cells. Both NKG2C⁺ and NKG2C^? NK cells proliferated in co‐culture with HCMV‐infected MRC‐5 cells, with the fraction of proliferating NKG2C⁺ NK cells directly correlating with the circulating NKG2C⁺ fraction. These data illustrate an at least partly NKG2C‐independent human NK‐cell memory‐type response against HCMV.     

Memory-like IFN-γ response by NK cells following malaria infection reveals the crucial role of T cells in NK cell activation by P. falciparum

NK cells are rapid IFN‐γ responders to Plasmodium falciparum‐infected erythrocytes (PfRBC) in vitro and are involved in controlling early parasitaemia in murine models, yet little is known about their contribution to immune responses following malaria infection in humans. Here, we studied the dynamics of and requirements for in vitro NK responses to PfRBC in malaria‐naïve volunteers undergoing a single experimental malaria infection under highly controlled circumstances, and in naturally exposed individuals. NK‐specific IFN‐γ responses to PfRBC following exposure resembled an immunological recall pattern and were tightly correlated with T‐cell responses. However, although PBMC depleted of CD56⁺ cells retained 20–55% of their total IFN‐γ response to PfRBC, depletion of CD3⁺ cells completely abrogated the ability of remaining PBMC, including NK cells, to produce IFN‐γ. Although NK responses to PfRBC were partially dependent on endogenous IL‐2 signaling and could be augmented by exogenous IL‐2 in whole PBMC populations, this factor alone was insufficient to rescue NK responses in the absence of T cells. Thus, NK cells make a significant contribution to total IFN‐γ production in response to PfRBC as a consequence of their dependency on (memory) T‐cell help, with likely positive implications for malaria vaccine development.     

NK cells modulate the lung dendritic cell‐mediated Th1/Th17 immunity during intracellular bacterial infection

The impact of the interaction between NK cells and lung dendritic cells (LDCs) on the outcome of respiratory infections is poorly understood. In this study, we investigated the effect and mechanism of NK cells on the function of LDCs during intracellular bacterial lung infection of Chlamydia muridarum in mice. We found that the naive mice receiving LDCs from C. muridarum‐infected NK‐cell‐depleted mice (NK‐LDCs) showed more serious body weight loss, bacterial burden, and pathology upon chlamydial challenge when compared with the recipients of LDCs from infected sham‐treated mice (NK+LDCs). Cytokine analysis of the local tissues of the former compared with the latter exhibited lower levels of Th1 (IFN‐γ) and Th17 (IL‐17), but higher levels of Th2 (IL‐4), cytokines. Consistently, NK‐LDCs were less efficient in directing C. muridarum‐specific Th1 and Th17 responses than NK+LDCs when cocultured with CD4⁺ T cells. In NK cell/LDC coculture experiments, the blockade of NKG2D receptor reduced the production of IL‐12p70, IL‐6, and IL‐23 by LDCs. The neutralization of IFN‐γ in the culture decreased the production of IL‐12p70 by LDCs, whereas the blockade of TNF‐α resulted in diminished IL‐6 production. Our findings demonstrate that NK cells modulate LDC function to elicit Th1/Th17 immunity during intracellular bacterial infection.     

PolyI:C plus IL‐2 or IL‐12 induce IFN‐γ production by human NK cells via autocrine IFN‐β

NK lymphocytes and type I IFN (IFN‐α/β) are major actors of the innate anti‐viral response that also influence adaptive immune responses. We evaluated type I IFN production by human NK cells in response to polyI:C, a potent type I IFN‐inducing TLR3 agonist. PolyI:C plus IL‐2/IL‐12 induced IFN‐β (but not IFN‐α) mRNA expression and protein production by highly pure human NK cells and by the human NK cell line NK92. Neutralizing anti‐IFNAR1 or anti‐IFN‐β Ab prevented the production of IFN‐γ induced by polyI:C plus IL‐2/IL‐12. Similarly, IFN‐γ production induced by polyI:C plus IL‐12 was reduced in NK cells isolated from IFNAR1^?/? compared with WT mice. The ability of polyI:C plus IL‐12 to induce IFN‐γ production was related to an increase of TLR3, Mda5 and IFNAR expression and by an increase of STAT1 and STAT4 phosphorylation. Collectively, these data demonstrate that NK cells, in response to polyI:C plus IL‐2/IL‐12, produce IFN‐β that induce, in an autocrine manner, the production of IFN‐γ and thereby highlight that NK cells may control the outcome of protective or injurious immune responses through type I IFN secretion.     

CCR6 and NK1.1 distinguish between IL‐17A and IFN‐γ‐producing γδ effector T cells

γδ T cells are a potent source of innate IL‐17A and IFN‐γ, and they acquire the capacity to produce these cytokines within the thymus. However, the precise stages and required signals that guide this differentiation are unclear. Here we show that the CD24^low CD44^high effector γδ T cells of the adult thymus are segregated into two lineages by the mutually exclusive expression of CCR6 and NK1.1. Only CCR6⁺ γδ T cells produced IL‐17A, while NK1.1⁺ γδ T cells were efficient producers of IFN‐γ but not of IL‐17A. Their effector phenotype correlated with loss of CCR9 expression, particularly among the NK1.1⁺ γδ T cells. Accordingly, both γδ T‐cell subsets were rare in gut‐associated lymphoid tissues, but abundant in peripheral lymphoid tissues. There, they provided IL‐17A and IFN‐γ in response to TCR‐specific and TCR‐independent stimuli. IL‐12 and IL‐18 induced IFN‐γ and IL‐23 induced IL‐17A production by NK1.1⁺ or CCR6⁺ γδ T cells, respectively. Importantly, we show that CCR6⁺ γδ T cells are more responsive to TCR stimulation than their NK1.1⁺ counterparts. In conclusion, our findings support the hypothesis that CCR6⁺ IL‐17A‐producing γδ T cells derive from less TCR‐dependent selection events than IFN‐γ‐producing NK1.1⁺ γδ T cells.     

NK cells exacerbate the pathology of influenza virus infection in mice

NK cells offer a first line of defense against viruses and are considered beneficial to the host during infection. Nevertheless, little is understood regarding the phenotype and function of NK cells in the lung during influenza virus infection. We found that the frequency of NK cells in mouse lung increased during influenza infection, with the majority of a mature phenotype. Cell surface CD107a and intracellular IFN‐γ were detected in cells expressing multiple NK‐cell receptors in infected lung, suggesting that NK cells were activated during infection. The activating receptor NKp46 was predominantly negative on such cells, possibly as a result of encountering influenza HA. Depletion of NK cells in vivo with anti‐asialo GM1 or anti‐NK1.1 reduced mortality from influenza infection and surviving mice recovered their body weight. Pathology induced by NK cells was only observed with high, not medium or low‐dose influenza infection, indicating that the severity of infection influences NK‐cell‐mediated pathology. Furthermore, adoptive transfer of NK cells from influenza‐infected lung, but not uninfected lung, resulted in more rapid weight loss and increased mortality of influenza‐infected mice. Our results indicate that during severe influenza infection of the lung, NK cells have a deleterious impact on the host, promoting mortality.     

CD2–CD58 interactions are pivotal for the activation and function of adaptive natural killer cells in human cytomegalovirus infection

The existence and expansion of adaptive NK‐cell subsets have been linked to HCMV infection. Phenotypically, a majority of adaptive NK cells expresses the activating receptor NKG2C and CD57. Some of the molecular factors driving the expansion of NKG2C⁺CD57⁺ NK cells in HCMV infection have been identified. The direct interaction of adaptive NK cells with HCMV‐infected cells, preceding the expansion, however, remains less studied. Recently, adaptive NK cells were reported to express higher levels of the co‐activating receptor CD2. We explored whether CD2 was directly involved in the response of adaptive NK cells to HCMV. In a co‐culture system of human PBMCs and productively infected fibroblasts, we observed an upregulation of CD69, CD25, and HLA‐DR on all NK cells. However, only in adaptive NK cells was this increase largely blocked by antibodies against CD2 and CD58. Functionally, this blockade also resulted in diminished production of IFN‐γ and TNF‐α by adaptive human NK cells in response to HCMV‐infected cells. Our results demonstrate that binding of CD2 to upregulated CD58 on infected cells is a critical event for antibody‐mediated activation and subsequent effector functions of adaptive NKG2C⁺CD57⁺ NK cells during the antiviral response.     

IFN‐γ directly inhibits murine B‐cell precursor leukemia‐initiating cell proliferation early in life

The early‐life immune environment has been implicated as a modulator of acute lymphoblastic leukemia (ALL) development in children, with infection being associated with significant changes in ALL risk. Furthermore, polymorphisms in several cytokine genes, including IL‐10 and IFN‐γ, are associated with leukemia development. However, the mechanisms and timing of these influences remain unknown. Here, we use the Eμ‐ret transgenic mouse model of B‐cell precursor ALL to assess the influence of IFN‐γ on the early‐life burden of leukemia‐initiating cells. The absence of IFN‐γ activity resulted in greater numbers of leukemia‐initiating cells early in life and was associated with accelerated leukemia onset. The leukemia‐initiating cells from IFN‐γ‐knockout mice had reduced suppressor of cytokine signaling (SOCS‐1) expression, were significantly more sensitive to IFN‐γ, and exhibited more rapid expansion in vivo than their wild‐type counterparts. However, sensitivity to this inhibitory pathway was lost in fully transformed IFN‐γ‐knockout leukemia cells. These results demonstrate that the influence of IFN‐γ on ALL progression may not be mediated by selection of nascent transformed cells but rather through a general SOCS‐mediated reduction in B‐cell precursor proliferation. Thus, while cytokine levels may influence leukemia at multiple points during disease progression, our study indicates a significant early influence of basal, infection‐independent cytokine production on leukemogenesis.     

IL‐27 stimulates human NK‐cell effector functions and primes NK cells for IL‐18 responsiveness

IL‐27, a member of the IL‐12 family of cytokines, is produced by APCs, and displays pro‐ and anti‐inflammatory effects. How IL‐27 affects human NK cells still remains unknown. In this study, we observed that mature DCs secreted IL‐27 and that blockade of IL‐27R (CD130) reduced the amount of IFN‐γ produced by NK cells during their coculture, showing the importance of IL‐27 during DC–NK‐cell crosstalk. Accordingly, human rIL‐27 stimulated IFN‐γ secretion by NK cells in a STAT1‐dependent manner, induced upregulation of CD25 and CD69 on NK cells, and displayed a synergistic effect with IL‐18. Preincubation experiments demonstrated that IL‐27 primed NK cells for IL‐18‐induced IFN‐γ secretion, which was associated with an IL‐27‐driven upregulation of T‐bet expression. Also, IL‐27 triggered NKp46‐dependent NK‐cell‐mediated cytotoxicity against Raji, T‐47D, and HCT116 cells, and IL‐18 enhanced this cytotoxic response. Such NK‐cell‐mediated cytotoxicity involved upregulation of perforin, granule exocytosis, and TRAIL‐mediated cytotoxicity but not Fas‐FasL interaction. Moreover, IL‐27 also potentiated Ab‐dependent cell‐mediated cytotoxicity against mAb‐coated target cells. Taken together, IL‐27 stimulates NK‐cell effector functions, which might be relevant in different physiological and pathological situations.     

Innate IFN‐γ promotes development of experimental autoimmune encephalomyelitis: A role for NK cells and M1 macrophages

The role of IFN‐γ in the pathogenesis of autoimmune diseases is controversial. Although Th1 cells can induce experimental autoimmune encephalomyelitis (EAE), IFN‐γ can suppress Th17 cells that are pathogenic in EAE. Here we show that NK cells provide an early source of IFN‐γ during development of EAE. Depletion of NK cells or neutralization of IFN‐γ delayed the onset of EAE and was associated with reduced infiltration of IL‐17⁺ and GM‐CSF⁺ T cells into the CNS. In the passive transfer model, immune cells from myelin oligodendrocyte glycoprotein (MOG)‐immunized IFN‐γ^?/? mice failed to induce EAE, despite producing IL‐17 and GM‐CSF. The macrophages expressed markers of M2 activation and the T cells had low very late antigen‐4 (VLA‐4) expression and failed to infiltrate the CNS. Addition of recombinant IFN‐γ to immune cells from the IFN‐γ^?/? mice activated M1 macrophages and restored VLA‐4 expression, migratory, and encephalitogenic activity of T cells. Furthermore, treatment of recipient mice with anti‐VLA‐4 neutralizing antibody abrogated EAE induced by transfer of T cells from WT mice. Our findings demonstrate IFN‐γ‐producing T cells are not required for development of EAE, but NK cell‐derived IFN‐γ has a key role in promoting M1 macrophage expansion and VLA‐4‐mediated migration of encephalitogenic T cells into the CNS.