Excessive inflammation is a postulated cause of severe disease and death in respiratory virus infections. In response to severe influenza virus infection, adoptively transferred naïve hemagglutinin-specific CD4+ T cells from CD4+ TCR-transgenic 6.5 mice drive an IFN-γ-producing Th1 response in wild-type mice. It helps in virus clearance but also causes collateral damage and disease aggravation. The donor 6.5 mice have all the CD4+ T cells with TCR specificity toward influenza hemagglutinin. Still, the infected 6.5 mice do not suffer from robust inflammation and grave outcome. The initial Th1 response wanes with time, and a prominent Th17 response of recent thymic emigrants alleviates inflammation and bestows protection in 6.5 mice. Our results suggest that viral neuraminidase-activated TGF-β of the Th1 cells guides the Th17 evolution, and IL-17 signaling through the non-canonical IL-17 receptor EGFR activates the scaffold protein TRAF4 more than TRAF6 during alleviation of lung inflammation in severe influenza.
Respiratory viruses cause serious emerging infectious diseases. The clinical outcome depends on the interplay between the invading virus and the host immune response. Viral invasion causes a cytopathic effect that leads to tissue injury in the respiratory tract. The host immune system reacts to clear the virus, but the response also contributes to inflammation. If the immune activation and inflammation abate with the virus clearance, the infected individual recovers efficiently from an infection. However, immune activation and inflammation may persist beyond the virus clearance. Prolonged and exaggerated immune activation causes aggravation of the disease. The undesirable outcome of seasonal or pandemic influenza virus infections, SARS, MERS, and the ongoing COVID-19 pandemics are examples1. Modulation of the inflammation with overwhelming immune reactions is always a goal in disease management. Corticosteroid as a commonly implemented agent to quell lung inflammation also impairs pathogen clearance with suppressed inflammation and may aggravate the disease, as seen in severe influenza2,3,4,5, SARS6, MERS7, and COVID-19 patients8.
Targeting cytokines is another option for suppressing inflammation. Tocilizumab and sarilumab target interleukin (IL)−6, and may help COVID-19 patients9. IL-17 is also a well-known pro-inflammatory cytokine10. The high IL-17 levels in patients with severe diseases of COVID-191,11,12, SARS13, MERS14, and influenza15, make IL-17 a possible target of manipulation16. We detected IL-17 in the sera of our patients with severe COVID-1911, and also in lung-infiltrating influenza antigen-specific CD4+ T cells in our experimental mouse model of severe influenza17,18.
In our influenza hemagglutinin (HA) antigen-specific mouse model, the influenza virus drives a Th1 response of adoptively transferred naïve HA-specific CD4+ T cells in the lungs17,18,19,20,21,22. The 2.5 × 106 adoptively transferred donor cells help in virus clearance but boost inflammation in the lungs and enhance mortality. Influenza virus infection of the donor mice, the HA-specific CD4+ T cell receptor (TCR)-transgenic 6.5 mice23, drives activation of all CD4+ T cells in these mice, as all their CD4+ T cells have the TCRs specific to the HA of the influenza virus. We expected a grave outcome with such a massive Th1 response of all CD4+ T cells in the infected 6.5 mice. Although they lost more body weight at first, they suffered from mild lung inflammation and recovered well. Their recovery was associated with a prominent Th17 response on days 6 and 9. Here we studied the role of Th17 response in protection of mice against severe influenza. We demonstrated a Th1 guided evolution of Th17 response. Our results suggest that viral neuraminidase-activated TGF-β of the Th1 cells guides the Th17 evolution. IL-17 signaling through the non-canonical IL-17 receptor EGFR activates the scaffold protein TRAF4 more than TRAF6 during alleviation of lung inflammation in severe influenza.
Th17 response is associated with the protection against severe influenza
In our influenza hemagglutinin (HA) antigen-specific mouse model17,18,19,20,21,22, wild-type mice received adoptive transfer of 2.5 × 106 naïve HA-specific 6.5 CD4+ T cells from 6.5 donor mice (WT mice + 6.5 cells) and got 2.5 × 103 plaque-forming units (p.f.u.) PR8 strain of H1N1 influenza virus infection. Lung-infiltrating HA-specific 6.5 CD4+ T cells (donor 6.5 cells) produced IFN-γ with the induction of Th1 transcription factor T-bet (Fig. 1a). There was base level IL-17 production with negligible Th17 transcription factor ROR-γt (Fig. 1a, Supplementary Note 1, Supplementary Fig. 1). Adoptive transfer aggravated the disease of wild-type mice with influenza virus infection. Lungs were inflamed (Fig. 1b), and lung-infiltrating donor 6.5 cells acquired effector function on day 6. These cells augmented the HA peptide-stimulated proliferation of cognate naïve CD4+ T cells in vitro (Fig. 1b). Virus burden also decreased in the lung from day 3 to day 9, as revealed in the plaque assay (Fig. 1c). The mice continued to lose body weight up to day 8, when some of them started to die (Fig. 1c).
The donor mice of the naïve HA-specific 6.5 CD4+ T cells, the 6.5 TCR transgenic mice, have monotonous TCR on their CD4+ T cells, with the same α/β TCR responding to an MHC Class II I-Ed restricted epitope of the HA from A/PR/8/34 (PR8) strain H1N1 influenza virus23. We expected a grave outcome of influenza virus infection in the donor 6.5 mice, with a massive Th1 response of all CD4+ T cells in the infected 6.5 mice. In contrast to our anticipation, the prominent Th1 response with IFN-γ production was noted on day 3 only, but not thereafter on days 6 and 9 after 2.5 × 103 p.f.u. PR8 virus infection (Fig. 1a). On days 6 and 9, with activation of Th17 transcription factor ROR-γt in 30 ~ 50% of the lung-infiltrating CD4+ T cells, 25 ~ 40% of them produced IL-17 instead (Fig. 1a, Supplementary Note 1, Supplementary Fig. 2). Infected 6.5 mice displayed alleviated lung inflammation (Fig. 1b), and their lung-infiltrating CD4+ T cells acquired regulatory function on day 6. These cells suppressed the HA peptide-stimulated proliferation of naïve cognate CD4+ T cells in vitro (Fig. 1b). Infected 6.5 mice lost significant body weight on day 3. They recovered from weight loss and all mice survived the infection (Fig. 1c). There was no live virus in the lungs on day 9 (Fig. 1c).
IL-17 (interleukin-17A) is a well-claimed pro-inflammatory cytokine which contributes to the pathogenesis of many inflammatory diseases11,12,13,14,15,16. But, a prominent IL-17 response of lung-infiltrating CD4+ T cells is associated with alleviated lung inflammation here in the infected 6.5 mice. The alleviation of lung inflammation and protection from influenza virus infection was lost in IL-17 knockout (KO) 6.5 mice. With 2.5 × 103 p.f.u. PR8 influenza virus, IL-17KO 6.5 mice incurred severe disease. The lung-infiltrating IL-17KO CD4+ and CD8+ T cells produced more inflammatory cytokines IFN-γ and TNF-α on day 6 (Fig. 1d).
Th17 is the response of recent thymic emigrants
Activated CD4+ T cells expand at first, followed by contraction of the pool of cells. Influenza virus-driven activation of the adoptively transferred naïve HA-specific 6.5 CD4+ T cells also followed a similar trend in infected wild-type mice (WT mice + 6.5 cells) during our experiments. The donor 6.5 cell pool expanded initially and then contracted in the lungs of wild-type mice (Fig. 2a). In contrast, the lung-infiltrating CD4+ T cell population decreased in the infected 6.5 mice in the first 4 days. It then started to increase and return to the level prior to infection by day 11 post-infection (Fig. 2a). Cells from the thymus of 6.5 mice, the new thymic emigrants, may replenish the population of lung-infiltrating CD4+ T cells. We thus tested CD24 and Qa2 expression profile of the lung-infiltrating CD4+ T cells in infected 6.5 mice. The matured thymocytes are known to express high-level CD24 and minimal Qa2. The CD4+ or CD8+ T cells lose CD24 and gain Qa2 expressions with time after being released to the periphery24. Among the lung-infiltrating CD4+ T cells of the infected 6.5 mice, we found a higher CD24 and lower Qa2 expressions on the surface of IL-17-producing than the IFN-γ-producing cells on day 6 (Fig. 2a).
To validate the above finding that suggests the recent thymic emigrants as the source of IL-17 in the lungs of infected 6.5 mice, we did thymectomy in 2-week-old 6.5 mice and infected the thymectomized 6.5 mice after 2 weeks of recovery from the surgery. Thymectomy is the surgical removal of the thymus gland. It thus abolishes the supply of thymic emigrants. After 2.5 × 103 p.f.u. PR8 influenza virus infection, thymectomized 6.5 mice had less ROR-γt induction and IL-17 production in the lung-infiltrating CD4+ T cells on day 7 (Fig. 2b). The proportions of IFN-γ-producing cells were not changed by thymectomy, and ~15% of the lung-infiltrating CD4+ T cells produced IFN-γ in the thymectomized and non-thymectomized 6.5 mice by this time (Fig. 2b). As a result, thymectomy obliterated the IL-17 dominance in infected 6.5 mice and reverted the distribution between IL-17 and IFN-γ back to the pattern similar to that of donor 6.5 cells in infected wild-type mice (Fig. 2b). With this altered Th response, thymectomy caused higher virus burden in the lungs (Fig. 2c), and aggravated disease with more bodyweight loss (Fig. 2d) in the infected 6.5 mice. These results corroborate our findings that suggest Th17 is a protective response of recent thymic emigrants in infected 6.5 mice.
Waning Th17 with aging-related thymic involution and severe disease of influenza virus infection in older than younger adult mice
Thymic involution causes decreased supply of thymic emigrants with age. Similar to wild-type mice, there were about 50 million thymocytes in 4-week-old 6.5 mice. The number decreased to 40 million in 8-week-old and 20 million in 12-week-old 6.5 mice. There were only 10 million thymocytes in 40-week-old 6.5 mice (Fig. 3a). In addition, there were less CD4+CD8+ double-positive cells in CD3+ thymocytes with age (Fig. 3b). Influenza virus infection induced less Th17 response in aged 6.5 mice compared to young 6.5 mice. About 30% of lung-infiltrating CD4+ T cells produced IL-17 in 4 or 8 week-old 6.5 mice on day 7 after the infection with 2.5 × 103 p.f.u. of PR8 influenza virus. The number decreased to less than 20% in 12-week-old and to around 10% in 40-week-old 6.5 mice (Fig. 3c, d). However, the percentage of IFN-γ producing cells did not change with age, and about 10–12% lung-infiltrating CD4+ T cells produced IFN-γ in the 4–40-week-old 6.5 mice (Fig. 3c, d). As the percentage of IFN-γ producing cells did not change with age, the IL-17 dominance over IFN-γ production also decreased with age (Fig. 3e). Virus burden in the lungs was higher in old than young 6.5 mice on day 7 after infection (Fig. 3f). Old 6.5 mice suffered from disease of more severity than young 6.5 mice, with delayed recovery of the weight loss (Fig. 3g).
The evolution of Th1-guided Th17 response
We devised an experiment with two adoptive transfers of naïve HA-specific 6.5 CD4+ T cells into infected wild-type mice (WT mice + 2-batches of 6.5 cells) to mimic sequential thymic emigrants in 6.5 mice (Fig. 4a). The donor 6.5 cells transferred with infection differentiated into IFN-γ-producing Th1 cells, and the donor 6.5 cells transferred 4 days later differentiated into IL-17-producing Th17 cells on day 8 after infection in the lungs of wild-type mice (Fig. 4a–c, Supplementary Note 2, Supplementary Fig. 3). In the presence of Th1 cells derived from first transfer, 30% of the second transfer cells produced IL-17 and only less than 2% produced IFN-γ. About 5% of IL-17-producing cells produced IFN-γ as well (Fig. 4d). The second transfer cells also modified the phenotype of first transfer cells. There was a 40% reduction of IFN-γ production on day 8 after infection (Fig. 4d). In single transfer controls (WT mice + single-batch 6.5 cells), the donor 6.5 cells transferred with infection or on days 4 post infection were prominent IFN-γ-producing Th1 cells on day 8 post infection in the lungs of wild-type mice. There was only a marginal decrease of IFN-γ production and a marginal increase of IL-17 production of the day 4 transferred cells compared to the cells transferred with infection. (Fig. 4).
In the experiment of wild-type mice with two adoptive transfers of naïve HA-specific 6.5 CD4+ T cells (WT mice + 2-batches of 6.5 cells), the IL-17-producing Th17 response of the second transfer donor 6.5 cells was associated with a decreased dominance of T-bet over ROR-γt (Fig. 5a, b). There were comparable TCR signal transduction from ZAP-70 phosphorylation to the induction of hallmark Th1 transcription factor T-bet in donor 6.5 cells of both transfers (Fig. 5c). With the substantial T-bet induction, there was a strong activation of Th17-associated transcription factor ROR-γt in the donor 6.5 cells of the second transfer (Fig. 5d). The donor 6.5 cells of the second transfer produced IL-17 instead of IFN-γ with the reduced T-bet dominance over ROR-γt (Fig. 5b–d). In addition, there was higher expression of the induced regulatory T (iTreg) cell marker LAG-3 in the donor 6.5 cells of the second transfer (Fig. 5e).
The two adoptive transfers of donor 6.5 cells helped the recipient wild-type mice to recover from the infection more efficiently with better survival, compared to the mice with one transfer of donor 6.5 cells only (Fig. 5f). Improved protection was there with less pathology (Fig. 5g), and lower viral load in the lungs on day 8 after infection (Fig. 5h).
We further defined the protective role of IL-17 from the second batch cells in the experiment with two adoptive transfers, by using donor 6.5 cells from naïve IL-17KO 6.5 mice in the second adoptive transfer ((WT mice + 1st batches 6.5 cells + 2nd batch IL-17KO 6.5 cells; Fig. 6a). Replacement of the second transfer cells with IL-17KO donor 6.5 cells abolished the protection with two transfers of IL-17 competent donor 6.5 cells (Fig. 6b). Despite facilitated virus clearance with the donor IL-17KO 6.5 cells (Fig. 6c), the mice suffered from aggravated disease with higher mortality with less IL-17 (Fig. 6d, f) and more inflammatory IFN-γ production in donor 6.5 cells of both transfers (Fig. 6e, f).
Influenza virus-activated TGF-β facilitates the evolution of Th17 response
The neuraminidase of the PR8 influenza virus activates TGF-β of the HA-specific CD4+ T cells18, and TGF-β is an indispensable cytokine for Th17 polarization in vitro25. Naïve HA-specific 6.5 CD4+ T cells were adoptively transferred into wild-type mice (WT mice + 6.5 cells) and the mice got infection of 2.5 × 103 p.f.u. of PR8 virus. The lung-infiltrating donor 6.5 cells had activated TGF-β on day 4 after infection in wild-type mice (Fig. 7a). They were retrieved from the recipient mice at that time for co-culture with naïve 6.5 CD4+ T cells in vitro. Without co-culture, the naïve 6.5 CD4+ T cells were Th1 cells and 16% of them produced IFN-γ after 48 h stimulation with PR8 virus in vitro. When co-cultured with the day 4 lung-infiltrating donor 6.5 cells, the naïve 6.5 CD4+ T cells were skewed to Th17 cells. Around 20% of them produced IL-17 and only 7% of them produced IFN-γ (Fig. 7b). Recombinant mouse TGF-β augmented and anti-TGF-β antibody attenuated the IL-17 production of the 6.5 CD4+ T cells (Fig. 7c). Vaccinia virus carrying HA (Vac-HA) activated adoptively transferred naïve HA-specific 6.5 CD4+ T cells in vivo to comparable amplitude as PR8 influenza virus. As Vac-HA does not have neuraminidase, donor 6.5 cells did not get activated TGF-β. After retrieval from the recipient mice on day 4, these Vac-HA activated lung-infiltrating donor 6.5 cells did not skew the Th1 cells into Th17 cells in the in vitro culture (Fig. 7).
Influenza virus-activated TGF-β of the Th1 cells guide naïve cells to respond with Th17 phenotype
In our earlier report, we demonstrated augmented TGF-β activation of the HA-specific CD4+ T cells in the absence of IL-1018. Here in, we tried two transfers of IL-10KO donor 6.5 cells into IL-10KO mice (IL-10KO mice + 2-batches of IL-10KO 6.5 cells; IL-10KO set), first transfer with PR8 influenza virus infection and second transfer on 4 days post infection. Two transfers of IL-10-competent donor 6.5 cells into wild-type mice (WT mice + 2-batches of 6.5 cells; WT set) served as controls (Fig. 8a, Supplementary Note 3, Supplementary Fig. 4). There was higher active-TGF-β in the IL-10KO donor 6.5 cells of the first transfer in IL-10KO setting with no IL-10, compared to the IL-10-competent donor 6.5 cells of the first transfer in WT setting with IL-10 (Fig. 8b). In addition, there was higher IL-17 in the IL-10KO donor 6.5 cells of the second transfer in IL-10KO setting with no IL-10, compared to the IL-10-competent donor 6.5 cells of second transfer in the WT setting with IL-10. About 60% of the second transfer cells produced IL-17 in the absence of IL-10 on day 8 post infection, compared to 25% in the setting with IL-10 (Fig. 8c), and the IL-17 production of the second transfer cells was linked to the protection against influenza virus infection in IL-10KO mice (Supplementary Note 3, Supplementary Fig. 4).
We next performed a similar two-transfer experiment in WT setting (WT mice + 2-batches of 6.5 cells) with HA-inserted vaccinia virus (Vac-HA) instead of PR8 influenza virus infection (Fig. 8d). The donor 6.5 cells of both transfers, transferred with infection and transferred on day 4, were activated into Th1 phenotype by the Vac-HA infection, as expected with the absence of active-TGF-β on the first transfer donor 6.5 cells (Fig. 8e).
IL-17 signaling through the non-canonical IL-17 receptor EGFR activates the scaffold protein TRAF4 more than TRAF6 in the lung-infiltrating HA-specific CD4+ T cells
IL-17 signaling through its well-known receptor IL-17RA contributes to inflammation26. Epidermal Growth Factor Receptor (EGFR; ErbB-1; HER1) has been described as a non-canonical receptor for IL-17. IL-17 signaling with EGFR has been demonstrated to participate in wound healing process27. We infected 6.5 mice with 2.5 × 103 p.f.u. of PR8 strain of influenza virus. Wild-type mice received adoptive transfer of naïve 6.5 donor cells with the infection (WT mice + 6.5 cells; WT set), and IL-17KO mice received adoptive transfer of naïve IL-17KO 6.5 donor cells with the infection (IL-17KO mice + IL-17KO 6.5 cells; IL-17KO set). There was up-regulation of both IL-17RA and EGFR on the lung-infiltrating CD4+ T cells in 6.5 mice after the influenza virus infection. IL-17RA was up-regulated only on day 3 post infection, but not thereafter. EGFR was up regulated gradually from day 3 to day 6 and day 9-post infection (Fig. 9a). EGFR was more abundant than IL-17RA on the cell surface from day 6 to day 9 post infection (Fig. 9b). Up-regulation of IL-17RA and EGFR were also there on lung-infiltrating donor 6.5 and IL-17KO 6.5 cells of WT set and IL-17KO set, respectively (Fig. 9a, b, Supplementary Note 4, Supplementary Fig. 5). However, there was EGFR dominance over IL-17RA on lung-infiltrating CD4+ T cell surfaces on day 6 but not on day 9 in WT set. There was no EGFR dominance over IL-17RA on days 6 and 9 in IL-17KO set (Fig. 9b).
Infected 6.5 mice had an abundance of EGFR on the surface of lung-infiltrating CD4+ T cells, and it was associated with higher phosphorylation of EGFR on day 9 than day 6 and day 3. In contrast, only a base level phosphorylated-EGFR was detected in the lung-infiltrating donor 6.5 cells of WT set and donor IL-17KO cells of IL-17KO set on days 3, 6, and 9 (Fig. 9c). Upon stimulation of splenocytes from naïve 6.5 mice, IL-17KO 6.5 mice, or WT mice with 0.03 MOI influenza virus for 48 h in vitro, there was more EGFR phosphorylation of 6.5 CD4+ T cells than that of IL-17KO 6.5 CD4+ T cells (Fig. 9d). IL-17 supplement augmented EGFR phosphorylation of IL-17KO 6.5 CD4+ T cells in a dose dependent manner, more augmentation with 1.0 than 0.1 μg/ml of recombinant mouse IL-17 (Fig. 9e). EGFR inhibitor Gefitinib mitigated EGFR phosphorylation of 6.5 CD4+ T cells and IL-17-induced EGFR phosphorylation of IL-17KO 6.5 CD4+ T cells (Fig. 9e). Wild-type CD4+ T cells from naïve WT mice served as controls, in which, IL-17 supplement augmented and Gefitinib attenuated EGFR phosphorylation (Fig. 9e).
Tumor Necrosis Factor Receptor-Associated Factors (TRAF) family (TRAF 1 to 7) of seven scaffold proteins are involved in various cellular physiological processes28. TRAFs, as adapter molecules, moderate many signaling pathways. TRAF4 and TRAF6 share the same TRAF binding sites, but they play different roles in the pathogenesis29,30. A real-time quantitative reverse transcription PCR study of Day 6 lung cells revealed up-regulation of TRAF4 and TRAF6 in the infected 6.5 mice. The TRAF4 was dominant over TRAF6 in the lung-infiltrating CD4+ T cells of infected 6.5 mice (Fig. 9f). In contrast, TRAF6 was dominant over TRAF4 in the lung-infiltrating donor 6.5 CD4+ T cells of infected wild-type (WT mice + 6.5 cells; WT set) or donor IL-17KO 6.5 CD4+ T cells of infected IL-17KO mice (IL-17KO mice + IL-17KO 6.5 cells; IL-17KO set; Fig. 9f). Upon similar in vitro stimulation, as described above, flow cytometry revealed more up-regulation of TRAF4 protein in 6.5 than IL-17KO 6.5 CD4+ T cells (Fig. 9g). IL-17 supplement augmented TRAF4 up-regulation in IL-17KO 6.5 CD4+ T cells in a dose dependent manner, more augmentation with 1.0 than 0.1 μg/ml of recombinant mouse IL-17 (Fig. 9g). EGFR inhibitor Gefitinib mitigated TRAF4 up-regulation in 6.5 CD4+ T cells and IL-17-induced TRAF4 up-regulation in IL-17KO 6.5 CD4+ T cells (Fig. 9g). Wild-type CD4+ T cells from naïve WT mice served as controls, in which, IL-17 supplement augmented and Gefitinib attenuated TRAF4 up-regulation (Fig. 9g).
Confocal microscopy revealed TRAF4 translocation to EGFR signaling complex in the 6.5 CD4+ T cells, upon stimulation of splenocytes from naïve 6.5 mice with 0.03 MOI influenza virus for 48 h in vitro (Fig. 9h). The co-localization of TRAF4 and EGFR was more significant in the 6.5 CD4+ T cells than that of IL-17KO 6.5 CD4+ T cells. IL-17 supplement augmented TRAF4 and EGFR co-localization in the stimulated IL-17KO 6.5 CD4+ T cells (Fig. 9h). EGFR inhibitor Gefitinib mitigated TRAF4 and EGFR co-localization in 6.5 CD4+ T cells and IL-17-induced TRAF4 and EGFR co-localization in IL-17KO 6.5 CD4+ T cells (Fig. 9h).
Treatment with EGFR inhibitor, Gefitinib, aggravates the disease of severe influenza
Gefitinib is an EGFR inhibitor, which interrupts signaling through the epidermal growth factor receptor (EGFR) in target cells. Gefitinib aggravated the disease of influenza virus infection in mice. The impact of EGFR inhibition with Gefitinib was highest in infected 6.5 mice, moderate in infected wild-type mice, and negligible in infected IL-17KO mice (Fig. 10a). In the infected wild-type mice, intranasal IL-17 supplement bestowed some protection, and the protection was lost with the treatment of Gefitinib (Fig. 10b). The lost benefit was associated with altered up-regulation of TRAF4 and TRAF6 in the lungs. The IL-17 supplement altered the TRAF6 dominance over TRAF4 in the lungs of infected wild type mice, and the alteration was restricted when the IL-17 supplement was given with Gefitinib treatment (Fig. 10b).
Here we demonstrated that influenza virus-activated TGF-β of the Th1 cells guides the Th17 evolution of recent thymic emigrants. IL-17 signaling through the non-canonical IL-17 receptor EGFR activates the scaffold protein TRAF4 more than TRAF6 during alleviation of lung inflammation in severe influenza. Adoptively transferred HA-specific 6.5 CD4+ T cells caused collateral damage with their IFN-γ-producing Th1 response to the infection in wild-type mice. The donor mice of the naïve HA-specific 6.5 CD4+ T cells, the 6.5 TCR transgenic mice, have all the CD4+ T cells responsive to the HA antigen of the influenza virus. The overwhelming CD4+ T cell response in the infected 6.5 mice would cause more damage and disease of more severity. There was aggravated illness at first, but the subsequent evolution of a prominent Th17 response mitigated the disease and the 6.5 mice had better survival than wild-type mice. Both Th1 and Th17 CD4+ T cells were activated for Th1 commitment with comparable TCR signaling from ZAP-70 phosphorylation to T-bet induction. The Th17 evolution was associated with ROR-γt upregulation and reduced T-bet dominance over ROR-γt. Thymectomy, aged mice experiments, and repetitive adoptive transfer experiments revealed the Th1 CD4+ T cells guided Th17 evolution, associated with viral neuraminidase activated TGF-β on the Th1 cells. IL-17 signaling through the EGFR happened with relative abundance of EGFR to IL-17RA in 6.5 CD4+ TCR-transgenic mice. The signaling activates the scaffold protein TRAF4 more than TRAF6. Gefitinib blockade documented the significance of IL-17 signaling through EGFR in alleviation of lung inflammation in severe influenza.
Both IFNγ and IL-17 are fundamental constituents of the cytokine storm in severe influenza with pneumonia31. IFN-γ is a hallmark Th1 cytokine essential for viral clearance32. In our experimental system, lung-infiltrating HA-specific CD4+ T cells produce IFN-γ in response to the infection17,18. The transcription factor T-bet directs commitment to the Th1 lineage and IFN-γ production33,34. Here we found our Th17 evolution was also preceded by Th1 commitment with a comparable strength of TCR signaling from ZAP-70 phosphorylation to T-bet induction. TGF-β is indispensable for Th17 polarization of naïve CD4+ T cells in vitro25. We revealed viral neuraminidase-activated TGF-β of the Th1 CD4+ T cells is essential for the Th1 guided evolution of Th17 cells. The Th1 CD4+ T cells steered activation of Th17-specific transcription factor ROR-γt and production of Th17-specific cytokine IL-17.
The Th1 transcription factor T-bet has been shown to suppress Th2 and Th17 differentiation35,36. However, co-production of IFN-γ and IL-17 has been detected in human CD4+ T cells25,37 and co-production IFN-γ and IL-10 has been documented in murine CD4+ T cells18. Relative expression levels of the transcription factors T-bet, GATA-3, and ROR-γt may fine tune a balance among different lineages of the conventional Th1/Th2/Th17 paradigm. A diminished T-bet dominance over GATA-3 and ROR-γt obliterates IFN-γ production of lung-infiltrating HA-specific CD4+ T cells in our experimental system17. Here, in the same experimental system of severe influenza, we demonstrated Th17 differentiation with diminished dominance of T-bet over ROR-γt. This excessive ROR-γt activation drove the Th1 committed CD4+ T cells into the Th17 lineage and abolish the IFN-γ production.
The role IL-17 remained uncertain. IL-17 is linked to localized inflammation and pathogenic in certain autoimmune diseases, in which IL-17 binds to its canonical receptor IL-17RA and activates TRAF6 and the subsequent signaling cascade26,28,38. IL-17 is anti-inflammatory in other diseases39,40,41. IL-17 is highly versatile and has been implicated in other non-pathogenic conditions including tissue repair and cancer as well42, in which IL-17 binds to recruited EGFR in the receptor complex and activates TRAF4 and subsequent signaling cascade27,39. TRAF4 activation may restrict IL-17-mediated pathogenic processes30. Our HA-specific Th17 cells were similar to the non-pathogenic Th17 cells25, as they do not produce IFN-γ and do not boost inflammation. There was also signaling through EGFR and subsequent activation of TRAF4. The Th17 response alleviated inflammation in our mouse model of severe influenza.
Aged people are at higher risk of severe influenza43. They also suffered from excessive mortality during SARS and the ongoing COVID-19 pandemics44,45, https://www.cdc.gov/coronavirus/2019-ncov/need-extra-precautions/index.html; accessed August 23, 2022. Their comorbidities contribute to this fact but it may also involve the derangement of the immune system. Age related thymic involution and decreased thymic output may be the problem. At 50 years of age, the human thymus generates only 15–20% of the T cells compared to the young. It is almost unable to make new T cells by the age of 65 (Ref: 46). Thymic involution starts from three months of age in mice47. The disease alleviating Th17 cells displayed the surface markers of recent thymic emigrants. Thymectomy abolished the Th17 response and the Th17 waned with age. The aged people may suffer from severe disease because of loss of the disease modifying Th17 cells. Our results raise alarms regarding modulating IL-17 response in severe respiratory tract infections. We have to be cautious to avoid inappropriate cytokine manipulation as IL-17 is not necessarily always proinflammatory.
Mice and influenza virus infection
All experiments involving mice were performed in accordance with protocols approved by the “Animal Care and Use Committee” of Chang Gung Memorial Hospital (Approval number: IACUC 2016030301). The committee certified that all experiments involving mice were carried out in strict accordance with the Animal Protection Law by the Council of Agriculture, Executive Yuan, Taiwan, R.O.C., and the guidelines contained in the Guide for the Care and Use of Laboratory Animals, as promulgated by the Institute of Laboratory Animal Resources, National Research Council, U.S.A.
All mice were of C57BL/10.D2 genetic background, and 8–12-week-old mice of both sexes, except in experiments with aged mice, were used for experiments. The TCR-transgenic mouse line 6.5 expresses a TCR recognizing an I-Ed-restricted HA epitope (110SFERFEIFPKE120) of HA of PR8 influenza virus (generously provided by H. Von Boehmer, Harvard University, Boston, MA). The 6.5 mice were crossed with syngeneic IL17KO or IL10KO mice to generate 6.5 IL17KO TCR-transgenic, or 6.5IL10 KO TCR-transgenic mice, respectively. Syngeneic IL17KO mice were generated by breeding IL17KO mice of B6 genetic background (generously provided by Y-C. Day, Chang Gung Memorial Hospital, Taiwan) into mice with a C57BL/10.D2 genetic background for >9 generations. Syngeneic IL10 KO and 6.5 IL10 KO TCR-transgenic mice were described earlier18. Mice were maintained under specific pathogen-free conditions.
The PR8 (A/PR/8/34) strain of influenza virus was produced in the allantoic fluid of 10-day-old embryonated chicken eggs, and characterized by a core facility at Chang Gung University, Taiwan. Mice were inoculated via the intranasal route during light isoflurane (Aesica Queenborough Ltd., Kent, UK) anesthesia with 2.5 × 103 plaque-forming units (p.f.u.s) in 50 μl of PBS17,18,19,20,21,22,34.
Infection with recombinant vaccinia virus encoding HA of PR8 strain of H1N1 influenza virus
IL-17 supplement & EGFR blockade
Recombinant mouse IL-17A (carrier free) was purchased from BioLegend (San Diego, CA, USA; Cat# 576006). An appropriate concentration of IL-17A in 20 μl PBS was supplemented daily through intranasal route for 5 days during isoflurane (Aesica Queenborough Ltd., Kent, UK) anesthesia.
The EGFR inhibitor Gefitinib was purchased from AstraZeneca. A stock solution in DMSO was first made and aliquots were kept in −80 °C. A fresh final solution was prepared every day in PBS and 100 mg/kg Gefitinib in 100 μl was fed by oral gavage.
Adoptive transfer and CFSE staining
Clonotypic HA-specific CD4+ TCR transgenic T cells were prepared from pooled spleens and lymph nodes of HA-specific CD4+ TCR-transgenic 6.5 mice17,18,19,20,21,22,34,48,49. Clonotypic IL17 KO or IL10 KO HA-specific CD4+ TCR-transgenic T cells were prepared from pooled spleens and lymph nodes of 6.5 IL17 KO or 6.5 IL10 KO TCR-transgenic mice, respectively. Clonotypic percentages were determined by flow cytometric analysis. Naïve phenotype was confirmed by assessing profiles of the activation markers CD44 (anti-mouse CD44; 553133, BD Biosciences; dilution = 1 in 100) and CD62L (anti-mouse CD62L; 553150, BD Biosciences; dilution = 1 in 100). Infection-responding 6.5 CD4+ T cells were retrieved from the lungs of infected HA-specific CD4+ TCR-transgenic 6.5 mice. CD4+ T cells were enriched by negative selection using magnetic beads, and donor cells were identified using fluorochrome-conjugated antibodies against CD90.1 (Thy1.1; anti-mouse CD90.1; 554898, BD Biosciences; dilution = 1 in 1000) or CD90.2 (Thy1.2; anti-mouse CD90.2; 553006; BD Biosciences; dilution = 1 in 200). For single-batch transfer experiments, 2.5 × 106 clonotypic 6.5 CD4+ T cells were injected via tail vein into Thy1.2/Thy1.2+ wild-type recipient mice in 0.2 ml of Hank’s Balanced Salt Solution (HBSS). For two-batch transfer experiments, a first batch of 0.5 × 106 clonotypic 6.5 CD4+ T cells was injected at the time of infection, and a second batch of 1.5 × 106 clonotypic 6.5 CD4+ T cells was injected on day 4 of infection in Thy1.2/Thy1.2+ wild-type mice.
Clonotypic T cells (1 × 107 cells/ml) were stained with 5 μM CFSE (5, 6-carboxyfluorescein diacetatesuccinimidyl ester; Molecular Probes, Eugene, OR, USA) in HBSS for 10 min. Cells were washed three times with HBSS, and CFSE-stained 6.5 CD4+ T cells (2.5 × 106) were transferred via tail vein into recipient mice.
Cell suspensions were incubated on ice with saturated concentrations of fluorochrome-labeled mAbs in FACS buffer (PBS plus 0.5% BSA and 0.02% NaN3) as recommended by the manufacturers. Donor CD4+ T cells were identified using the following mAbs: biotin-conjugated anti-clonotypic 6.5 TCR (provided by H. Von Boehmer; dilution = 1 in 100), avidin-PE (A1204, Caltag, Burlingame, CA; dilution = 1 in 200), or Streptavidin-APC (SA 1005, Caltag, Burlingame, CA; dilution = 1 in 200), and PerCP or FITC-conjugated anti-CD4 (553052 or 553047, BD Biosciences; dilutions = 1 in 100 for both). All fluorochrome-conjugated antibodies were from BD Biosciences, except for T-bet, and mouse IgG1κ isotype control (45–5825, 45–4714, eBiosciences; dilution = 1 in 50 for both), FITC-Phospho-EGFR (Tyr1068) Rabbit anti-Human, Mouse (MA5-27995, Invitrogen; dilution = 1 in 10), and PE-TRAF4 (SC-390232, Santa Cruz Biotechnology; no dilution). Cells were acquired using FACS CALIBUR, and analysis was performed using CellQuest Pro (BD Biosciences), FACSuite (BD Biosciences), or FlowJo (Tree Star, Inc.) software.
Intracellular cytokine staining
Single cell suspensions (5–10 × 106 cells well−1 in 24 well plates) from harvested organs were re-stimulated for 5 h with 10 μg ml−1 MHC class II restricted HA peptide (SFERFEIFPKE) in the presence of 5 μg ml−1 Brefeldin A (SI-B7651, Sigma-Aldrich). The Brefeldin-A concentration was maintained throughout intracellular cytokine staining. Re-stimulated cells were surface-stained with anti-CD90.1 and anti-CD4 as described above, fixed in IC fixation buffer (00–8222, eBiosciences), washed, and stained in permeabilization buffer (00–8333, eBiosciences) containing fluorochrome-conjugated antibodies against target cytokines. For Foxp3 (11–5773, eBiosciences; dilution = 1 in 50) and T-bet, cells were surface-stained, fixed, and permeabilized in fixation/permeabilization buffer (00-5523, eBiosciences), washed, and stained in permeabilization buffer (00-8333, eBiosciences) containing fluorochrome-conjugated antibodies. In some experiments, cells were analyzed ex vivo and stained without re-stimulation.
In vitro proliferation of naïve HA-specific CD4+ T cells
Single cell suspensions were prepared from the spleens of HA-specific TRC transgenic 6.5 mice. Splenocytes (2.5 × 105) were re-stimulated for 72 h with class-II restricted HA peptide (SFERFEIFPKE) at 37 °C in the presence of 5% CO2. Tritiated thymidine was added 18 h prior to harvest. The amount of radioactivity incorporated into DNA in each well was measured in a scintillation counter, as described earlier17,34. We co-cultured lung-infiltrating HA-specific CD4+ T cells retrieved from infected mice as described in the text, to measure their impact on proliferation.
Cytokine production and expression of TRAF4 and p-EGFR following in vitro stimulation
For cytokine production, single-cell suspensions of splenocytes (2.5 × 106 cells per well) from IL-17-competent (IL-17+/+) 6.5 mice were stimulated with influenza virus (PR8 strain; 0.03 (MOI) Multiplicity of Infection) in complete RPMI-1640 for 48 h in vitro, in the presence of lung-infiltrating HA-specific 6.5 CD4+ T cells from day 4 infected mice at 1:5 ratio. Naïve splenocytes without cells from the infected mice served as controls. Recombinant mouse TGF-β (10 ng ml−1, GF346, Millipore) or anti- TGF-β (10 μgml−1, MAB1835, R&D) was added to the culture at the onset. Cognate HA peptide (100 μgml−1) was added to the culture for last 5 h. Brefeldin-A was added for last 3 h of stimulation and cells were then processed for intracellular staining.
For TRAF4 staining, single-cell suspensions of splenocytes (2.5 × 106 cells per well) from naive WT, IL-17-competent (IL-17+/+) 6.5, or IL-17 knockout (IL-17−/−) 6.5 mice were stimulated with influenza virus (PR8 strain; 0.03 (MOI) Multiplicity of Infection) in complete RPMI-1640 for 48 h in vitro. Recombinant mouse IL-17 (0.1 or 1.0 μgml−1; 576006, Biolegend) and/or Gefitinib (0.1 or 1.0 μgM; Iressa, AstraZeneca) was added to the culture at the onset. Cognate HA peptide (100 μgml−1) was added to the culture for last 5 h. Brefeldin-A was added for last 3 h of stimulation and cells were then processed similarly, as we did for intracellular staining.
For phosphorylated-EGFR staining, single-cell suspensions of splenocytes (2.5 × 106 cells per well) from naive WT, IL-17-competent (IL-17+/+) 6.5, or IL-17 knockout (IL-17−/−) 6.5 mice were stimulated with influenza virus (PR8 strain; 0.03 (MOI) Multiplicity of Infection) in complete RPMI-1640 for 48 h in vitro. Recombinant mouse IL-17 (0.1 or 1.0 μgml−1; 576006, Biolegend) and/or Gefitinib (0.1 or 1.0 μgM; Iressa, AstraZeneca) was added to the culture at the onset. Cognate HA peptide (100 μgml−1) was added to the culture for last 5 h. Brefeldin-A was added for last 3 h of stimulation and cells were then processed for staining. In brief, cells were first placed in cold perm buffer-III (558050, BD Biosciences) for 30 min, washed with stain buffer (554657, BD Biosciences), and stained with FITC-Phospho-EGFR (Tyr1068) Rabbit anti-Human, Mouse (MA5-27995, Invitrogen; dilution = 1 in 10) in stain buffer. Cells were acquired with BD LSRFortessa™ Cell Analyzer for flow cytometry or visualized in confocal microscopy (Leica).
Plaque assay for virus titer
We measured live influenza virus titers in the organs of infected mice using a modified Madin-Darby canine kidney cell (MDCK; The American Type Culture Collection) plaque assay, as described earlier17,18,19,20,21,22,34. The organs were collected at the indicated times in 1 mL DMEM, snap-frozen in liquid nitrogen, and stored at −80 °C until analysis. Ten-fold dilutions of tissue homogenates were prepared in DMEM supplemented with 10% FCS, antibiotic-antimycotic (15240-062, GIBCO BRL), and 0.00025% trypsin. A total of 500 μl of each dilution was added to confluent monolayers of MDCK cells in 6 well plates and incubated for 1 h at 35 °C with 5% CO2. Each well then received 2 mL of agar overlay (0.3%) and was incubated for 3 days. Cells were fixed with 10% formalin, agar overlay was removed, and fixed monolayers were stained with crystal violet (0.02% in 2% ethanol). The results were presented as plaque forming unit (PFU)/ml = (mean number of plaques × 2) × (dilution factor−1).
The lungs from experimental mice were harvested on day 7 post-infection, and fixed with 10% neutral buffered formalin solution. Following fixation, lungs were embedded in paraffin and 5 μm sections were cut. Sections were stained with hematoxylin and eosin (H&E) and scored blindly. Infiltration of inflammatory cells, including lymphocytes, neutrophils, and plasma cells, was separately scored by pathologists as negative, 1+, 2+, or 3+ according to the density of infiltration. Vasculitis and fibrosis were also scored as negative, 1+, 2+, or 3+ based on severity, as described earlier17,18,19,20,21,22,34. Overall inflammation in the lungs was represented by the average of these scores.
Statistics and reproducibility
Data are expressed as mean ± s.d. We used Graphpad Prism version 8 for Student’s t test analyses to compare two groups at a specific time point. We also analyzed Two-way ANOVA for the body weight curves and Gehan–Breslow–Wilcoxon test for survival curves. We considered p values < 0.05 as significant.
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
All data supporting the findings of this study are available within the paper and its Supplementary Information. The numerical source data for the graphs can be found in Supplemental Data 1.
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We thank the Radiation Research Core Laboratory of the Chang Gung Memorial Hospital for the flow cytometry instrument support. The Research Center for Emerging Viral Infections receives support from The Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE), Taiwan and NSTC 111−2634-F-182-001 from the National Science and Technology Council, Taiwan. This work was supported by Projects MOST 111-2314-B-182-029 (A.D.), MOST-105-2321-B-182A-003-MY3 (A.D.), and MOST-106-2314-B-182A-160-MY3 (C.T.H.) from the Ministry of Science & Technology, Taiwan, and by CMRPVVJ0052 (C.Y.H.), CMRPG 3E2081-3, and 3J0721 (C.T.H.) from the Medical Research Project Fund, Chang Gung Memorial Hospital, Taiwan.
The authors declare no competing interests.
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Dutta, A., Hung, CY., Chen, TC. et al. An IL-17-EGFR-TRAF4 axis contributes to the alleviation of lung inflammation in severe influenza. Commun Biol 6, 600 (2023). https://doi.org/10.1038/s42003-023-04982-0