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. 2020 Feb 5;16(2):e1008279.
doi: 10.1371/journal.ppat.1008279. eCollection 2020 Feb.

Selective reconstitution of IFN‑γ gene function in Ncr1+ NK cells is sufficient to control systemic vaccinia virus infection

Katharina Borst  1 , Sven Flindt  2 , Patrick Blank  1 , Pia-Katharina Larsen  1 , Chintan Chhatbar  1 , Jennifer Skerra  1 , Julia Spanier  1 , Christoph Hirche  1 , Martin König  2 , Tomas Alanentalo  3 , Martin Hafner  4 , Zoe Waibler  2 , Klaus Pfeffer  5 , Veronika Sexl  6 , Gerd Sutter  7 , Werner Müller  4 , Theresa Graalmann  1   8 , Ulrich Kalinke  1   9
Affiliations

Selective reconstitution of IFN‑γ gene function in Ncr1+ NK cells is sufficient to control systemic vaccinia virus infection

Katharina Borst et al. PLoS Pathog. .

Abstract

IFN-γ is an enigmatic cytokine that shows direct anti-viral effects, confers upregulation of MHC-II and other components relevant for antigen presentation, and that adjusts the composition and balance of complex cytokine responses. It is produced during immune responses by innate as well as adaptive immune cells and can critically affect the course and outcome of infectious diseases, autoimmunity, and cancer. To selectively analyze the function of innate immune cell-derived IFN-γ, we generated conditional IFN-γOFF mice, in which endogenous IFN-γ expression is disrupted by a loxP flanked gene trap cassette inserted into the first intron of the IFN-γ gene. IFN-γOFF mice were intercrossed with Ncr1-Cre or CD4-Cre mice that express Cre mainly in NK cells (IFN-γNcr1-ON mice) or T cells (IFN-γCD4-ON mice), respectively. Rosa26RFP reporter mice intercrossed with Ncr1-Cre mice showed selective RFP expression in more than 80% of the NK cells, while upon intercrossing with CD4-Cre mice abundant RFP expression was detected in T cells, but also to a minor extent in other immune cell subsets. Previous studies showed that IFN-γ expression is needed to promote survival of vaccinia virus (VACV) infection. Interestingly, during VACV infection of wild type and IFN-γCD4-ON mice two waves of serum IFN-γ were induced that peaked on day 1 and day 3/4 after infection. Similarly, VACV infected IFN-γNcr1-ON mice mounted two waves of IFN-γ responses, of which the first one was moderately and the second one profoundly reduced when compared with WT mice. Furthermore, IFN-γNcr1-ON as well as IFN-γCD4-ON mice survived VACV infection, whereas IFN-γOFF mice did not. As expected, ex vivo analysis of splenocytes derived from VACV infected IFN-γNcr1-ON mice showed IFN-γ expression in NK cells, but not T cells, whereas IFN-γOFF mice showed IFN-γ expression neither in NK cells nor T cells. VACV infected IFN-γNcr1-ON mice mounted normal cytokine responses, restored neutrophil accumulation, and showed normal myeloid cell distribution in blood and spleen. Additionally, in these mice normal MHC-II expression was detected on peripheral macrophages, whereas IFN-γOFF mice did not show MHC-II expression on such cells. In conclusion, upon VACV infection Ncr1 positive cells including NK cells mount two waves of early IFN-γ responses that are sufficient to promote the induction of protective anti-viral immunity.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Cell selectivity of Cre-mediated recombination using CD4-Cre and Ncr1-Cre mice.
(A) Schematic depiction of TRAP-mediated inactivation of IFN-γ and of Cre-mediated reconstitution of the Ifng gene function. (B) Splenocytes of R26RFPOFF (white) and R26RFPCD4-ON mice (black) were isolated and immune cell subsets were analyzed by flow cytometry for RFP expression (n = 3, N = 1), one-tailed Mann-Whitney U test. (C) Splenocytes of R26RFPOFF (white) and R26RFPNcr1-ON mice (black) were isolated and T and NK cells were analyzed by flow cytometry for RFP expression (n ≥ 3, N = 2), one-tailed Mann-Whitney U test. (D) Splenocytes were isolated from WT, IFN-γOFF and IFN-γCD4-ON mice, stimulated with PMA/ionomycin for 4 h, and then analyzed by flow cytometry (n ≥ 3, N = 2), paired T-test. Percentage of IFN-γ expressing T or NK cells is shown. Error bars indicate mean ± SEM; ***p ≤ 0.001. (E) Splenocytes were isolated from WT, IFN-γOFF and IFN-γNcr1-ON mice, stimulated with PMA/ionomycin for 4 h, and then analyzed by flow cytometry (n ≥ 3, N = 1–3), paired T-test. Percentage of IFN-γ expressing T or NK cells is shown. Error bars indicate mean ± SEM; ***p ≤ 0.001. (F) Immune cells were isolated from naïve WT, IFN-γOFF and IFN-γNcr1-ON mice and total numbers of T cells, B cells, macrophages, and polymorphonuclear neutrophils (PMN) in spleen was analyzed by FACS (n ≥ 9, N = 3), one-tailed Mann-Whitney U test.
Fig 2
Fig 2. IFN-γNcr1-ON mice are protected against lethal VACV infection.
WT, IFN-γOFF, IFN-γCD4-ON and IFN-γNcr1-ON mice were i.v. infected with 2 x 106 pfu VACV. (A) Serum samples were drawn at the indicated time points and analyzed for the IFN-γ content by an ELISA method (n = 6, N = 2); one-way Anova. (B) Survival was monitored and in case body weight decrease by more than 20% of the initial bodyweight, or when the overall health status was dramatically reduced, mice were sacrificed (n ≥ 10, N = 3); Mantel Cox test. (C) Virus loads in ovaries was determined 5 days post infection (dpi) by plaque assay (n ≥ 5, N = 2). Error bars indicate mean ± SEM; ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05; one-tailed Mann-Whitney U test.
Fig 3
Fig 3. Balanced cytokine responses in VACV infected IFN-γNcr1-ON mice.
WT, IFN-γOFF and IFN-γNcr1-ON mice were i.v. infected with 2 x 106 pfu VACV. (A/B) Splenocytes of infected mice were isolated 1 and 4 dpi, in vitro stimulated with CD3/CD28 beads for 4 h and then analyzed by flow cytometry for IFN-γ expressing NK or T cells (n ≥ 10, N = 3); one-tailed Mann-Whitney U test. Error bars indicate mean ± SEM; ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05. (C) Serum samples were drawn at the indicated time points and analyzed with a multiplex cytokine and chemokine array (n = 3, N = 1).
Fig 4
Fig 4. Normalized distribution of peripheral myeloid cell subsets in VACV infected IFN-γNcr1-ON mice.
WT, IFN-γOFF and IFN-γNcr1-ON mice were i.v. infected with 2 x 106 pfu VACV. At the indicated time points blood samples were drawn, spleen was prepared and myeloid cells were analyzed by flow cytometry (n ≥ 7, N = 3). (A) Percentages and total cell numbers of polymorphonuclear neutrophils (PMN) in the blood. Percentages and total cell numbers of (B) PMN or (C) macrophages in spleen (pregated on CD3-, CD19- cells). (D) Percentages of MHC-II expressing macrophages in the spleen.
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This study was supported by funding from the Helmholtz Association (Zukunftsthema “Immunology & Inflammation” (ZT-0027); https://www.helmholtz.de/en/), funding from the Deutsche Forschungsgemeinschaft (Joint French-German Project cGAS-VAC, project number 406922110; http://www.dfg.de/), by INVADERS (Innate immunity and vaccine development: role of soluble mediators), Grant number: QLK2-CT-2001-02103, (https://cordis.europa.eu/project/rcn/60038_en.html) to U.K.; the International Training Group IRTG1273 funded by the German Research Foundation (DFG) (https://www.mh-hannover.de/4587.html) to K.B. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.