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Type I interferon regulates proteolysis by macrophages to prevent immunopathology following viral infection [1]

['Amanda J. Lee', 'Department Of Medicine', 'Mcmaster Immunology Research Centre', 'Mcmaster University', 'Hamilton', 'Ontario', 'Emily Feng', 'Marianne V. Chew', 'Elizabeth Balint', 'Sophie M. Poznanski']

Date: 2022-07

The ability to treat severe viral infections is limited by our understanding of the mechanisms behind virus-induced immunopathology. While the role of type I interferons (IFNs) in early control of viral replication is clear, less is known about how IFNs can regulate the development of immunopathology and affect disease outcomes. Here, we report that absence of type I IFN receptor (IFNAR) is associated with extensive immunopathology following mucosal viral infection. This pathology occurred independent of viral load or type II immunity but required the presence of macrophages and IL-6. The depletion of macrophages and inhibition of IL-6 signaling significantly abrogated immunopathology. Tissue destruction was mediated by macrophage-derived matrix metalloproteinases (MMPs), as MMP inhibition by doxycycline and Ro 28–2653 reduced the severity of tissue pathology. Analysis of post-mortem COVID-19 patient lungs also displayed significant upregulation of the expression of MMPs and accumulation of macrophages. Overall, we demonstrate that IFNs inhibit macrophage-mediated MMP production to prevent virus-induced immunopathology and uncover MMPs as a therapeutic target towards viral infections.

Dysregulated immune responses and their associated pathologies are the culprit of severe disease symptoms in response to viral infections. The ability to properly regulate effective and controlled immune responses is a critical feature of preventing severe disease outcomes. Type I interferons are antiviral signaling molecules known to induce potent antiviral immune responses; however, their ability to suppress pathogenic immune responses is poorly understood. Employing a vaginal HSV-2 infection model in mice, we show that type I IFN signaling is critical to preventing the development of severe tissue pathology by suppressing the pathogenic functions of macrophages. In the absence of type I IFNs, these unleashed macrophages produce MMPs that can degrade tissue structure. We show that inhibiting MMPs reduces the severity of immunopathology. We further provide evidence that influenza infection in mice, as well as severe COVID-19 infection in humans, is linked to macrophage and MMP-mediated tissue destruction. Together, our study describes a distinct mechanism through which type I IFNs regulate pathogenic immune responses, and defines MMPs as a potential therapeutic target during severe viral infections.

Funding: This work was funded by the Canadian Institutes of Health Research (CIHR) (20008285 to A.A.A). A.A.A holds a Tier 1 Canadian Research Chair in Natural Immunity and NK Cell Function. A.J.L and S.M.P are recipients of a CIHR Vanier Canada Graduate Scholarship. E.F and E.B, are supported by a Master’s Canadian Graduate Scholarship and Master’s Ontario Graduate Scholarship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Here, we report that in the absence of IFNAR, mucosal viral infection leads to the development of significant immune-mediated tissue pathology in mice. We found that type I IFN signaling suppressed IL-6 production. In the absence of IFN-mediated suppression, IL-6 was critical in inciting immunopathology through the induction of phagocytic macrophages and their production of matrix metalloproteinases (MMPs). Interestingly, direct inhibition of MMP proteolytic activity was sufficient to limit immunopathology during infection. Further, we show that the immunoregulatory function of type I IFNs occurs independent of the location and type of viral infection. Overall, we define a previously unidentified immunomodulatory function of type I IFN, which acts as a suppressor of IL-6-induced macrophage and MMP-mediated immunopathology to prevent tissue damage and increase disease tolerance. We further identify MMPs as a promising therapeutic target in limiting damage and promoting survival following mucosal viral infections.

The immune-mediated tissue pathology during viral infection is fueled by pro-inflammatory cytokines such as IL-6, TNF-α and IL-1β [ 12 ]. These responses have been recognised in not just infectious diseases, but also in graft-versus-host disease and various chronic inflammatory disorders [ 13 – 15 ]. Of these cytokines, IL-6 signaling has been particularly emphasized for its ability to regulate various aspects of both protective and pathogenic immune responses. Elevated IL-6 has been specifically highlighted as a contributor to pathogenesis during severe COVID-19 and IAV, and many efforts to block IL-6 signaling to improve disease outcomes are underway [ 16 – 19 ]. Inhibiting IL-6 responses can impair leukocyte recruitment and dampen T cell and neutrophil antiviral responses [ 20 – 24 ]. Furthermore, IL-6 has also been described as the driving force behind CRS [ 25 ]. However, despite the well-accepted relationship between IL-6 and inflammation, the specific role of IL-6 in the development of immune-mediated tissue pathology is poorly defined. We hypothesize that type I IFNs play a central role in suppressing cytokine-driven hyperinflammation during infection.

Studies of highly pathogenic viral infections, including severe acute respiratory syndrome coronavirus type 2 (SARS-CoV)-2 and Ebola virus (EBOV), demonstrate that early type I IFN induction consistently correlates with disease tolerance [ 5 – 7 ]. These immunopathogenic responses often occur independently of viral load and are the result of dysregulated heightened inflammation [ 8 ]. Type I IFNs have demonstrated an ability to control potentially pathogenic immune responses. During HSV-2 infection, type I IFNs both induce IFN-γ production by NK cells, but also inhibit their production during later stages of infection [ 9 ]. In a mouse model of influenza infection, type I IFNs have also been demonstrated to inhibit various sources of immune-mediated pathology [ 10 , 11 ]. Understanding how type I IFNs can both promote viral clearance, but also control inflammation to improve disease outcomes, is critical to understanding the mechanisms of disease pathology and develop effective treatments for viral infections.

Effective early innate immune responses are crucial in determining the clinical outcomes of viral infections. Dysregulated inflammatory immune responses will not only impede early viral replication, but ultimately lead to immune-mediated tissue pathology. Type I interferons (IFNs) are master regulators of the early innate immune response and determinants of disease outcome against viral infection [ 1 , 2 ]. For instance, during genital herpes simplex virus type 2 (HSV-2) infections, type I IFN induction stimulates early antiviral immunity through promoting monocyte recruitment and Natural Killer (NK) cell production of IFN-γ, necessary for host defense [ 3 ]. The absence of type I IFN signaling results in increased viral replication and reduced survival to vaginal HSV-2 infection [ 3 , 4 ].

Results

Vaginal immunopathology in Ifnar-/- mice is independent of ILC2s or TH2 CD4+ T cells It has been shown that deficiency or absence of type I IFN signaling is associated with an increased inflammatory response, and reduced type I IFN induction is correlated with disease severity in COVID-19 patients [7,29]. However, how type I IFN signaling prevents excessive inflammation and tissue damage during viral infections is less clear. Some have speculated that type I IFNs can suppress inflammatory responses mediated by TH2 cells and group 2 innate lymphoid cells (ILC2s) in response to allergens or infections [11,30]. To assess the involvement of any lymphoid-derived cells in vaginal immunopathology, we employed NOD-Rag2-/-IL2rγ-/- (NRG) mice lacking any lymphoid lineage cells, including ILC2s and CD4+ T-cells. Blocking IFNAR signaling through administering an α-IFNAR antibody in NRG mice, like C57BL/6 Ifnar-/- mice, was sufficient to induce significant vaginal immunopathology and loss of collagen staining, compared to isotype-matched Ig controls (Fig 2A–2C). To confirm these findings in Ifnar-/- mice, we detected only a modest increase in proportion of ILC2 cells between WT and Ifnar-/- mice at 2 dpi, and no difference in the total number of ILC2 cells between the two groups (Figs 2D, 2E and S2A). We also detected no differences in the levels of vaginal IL-33, a potent stimulator of ILC2 cells, between WT and Ifnar-/- mice 1 dpi (S2B–S2D Fig). WT and Ifnar-/- mice also had comparable levels of CD3+ T cells, both in proportion and total number, in their vaginal mucosa at 2 and 3 dpi (Figs 2F, 2G and S2E). Furthermore, depletion of CD4+ T cells through administering an α-CD4 depleting antibody in Ifnar-/- mice resulted in no observable difference in the degree of immunopathology compared to control mice (S2F–S2J Fig). These experiments collectively indicate that virus-induced vaginal immunopathology in the absence of IFNAR occurs independently of lymphoid lineage cells, including both ILC2s and CD4+ T-cells, as well as NK cells and CD8+ T cells. PPT PowerPoint slide

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TIFF original image Download: Fig 2. Vaginal immunopathology in Ifnar-/- mice is independent of ILC2s or TH2 CD4+ T cells. (A) H&E staining of vaginal cross-sections of NRG mice + α-IFNAR or isotype control 3 dpi (n = 5). (B and C) PSR staining (B) and quantification of PSR+ to total vaginal tissue (C) of NRG mice + α-IFNAR or isotype control 3 dpi (n = 5). (D and E) Total number of CD45+Lin-ST2+CD90.2+ ILC2s (D) and proportion of ILC2s to total CD45+ cells (E) in vaginal tissue of WT and Ifnar-/- mice following HSV-2 infection (n = 3). (F and G) Total number of CD3+ T cells (F) and proportion of CD3+T cells to total CD45+ cells (G) in vaginal tissue (n = 3). 10x scale bar represents 200 μm, 20x and PSR scale bar represents 100 μm. Data in (C) to (D), (E), (F), and (G), are represented as mean ± SEM. *p < 0.05. (D-G, two-way ANOVA; C two-tailed t test) See also S2 Fig. https://doi.org/10.1371/journal.ppat.1010471.g002

Phagocytic cells are required for HSV-2-induced tissue immunopathology in Ifnar-/- mice Since we observed that neither ILCs nor a lymphoid cell-mediated type II immune response were responsible for vaginal pathology in the absence of type I IFN signaling, we addressed whether immunopathology was induced by innate immune cells of the myeloid lineage. We examined the influx of neutrophils and eosinophils following infection of the vaginal mucosa of WT and Ifnar-/- mice. We detected a significant increase in the proportion and total number of neutrophils in the vaginal mucosa at 2 dpi in Ifnar-/- mice compared to WT mice (S3A–S3C Fig). To determine whether the increase in neutrophils in Ifnar-/- mice was responsible for HSV-2-induced tissue pathology, we depleted neutrophils using the α-Ly6G mAb in Ifnar-/- mice. There was significant depletion of neutrophils at 3 dpi in the blood, spleen, and vaginal tissue of WT mice (S3D–S3F Fig). However, α-Ly6G administration did not abrogate vaginal tissue destruction and collagen degradation in Ifnar-/- mice (S3G–S3l Fig). Thus, while neutrophil recruitment is increased in the absence of IFNAR, they are not required for vaginal tissue pathology. We also examined eosinophil populations in the vaginal mucosa and observed no difference in the proportion or total cell numbers between WT and Ifnar-/- mice, suggesting that eosinophils are also not involved in vaginal immunopathology (S3J–S3L Fig). In instances of virus-induced lung immunopathology, macrophages and inflammatory monocytes (IM) have been implicated in the immunopathological process through the release of pro-inflammatory cytokines and the upregulation of TRAIL [31–33]. We have previously described that infiltration of IMs to HSV-2-infected vaginal tissue is impaired in Ifnar-/- mice (3). To determine if there were changes in macrophage populations, we examined levels of F4/80+ cells at 0 and 3 dpi in the vaginal tissue in WT and Ifnar-/- mice. Though we did not detect a difference in the proportion of macrophages, we found a significant increase in the total number of vaginal F4/80+ cells in Ifnar-/- mice at 3 dpi (Figs 3A, 3B and S3M). To investigate if macrophages contribute to tissue pathology, clodronate liposomes were administered to reduce the number of phagocytic macrophages (Figs 3C and S3N). Clodronate administration in Ifnar-/- mice markedly reduced tissue damage without impeding control of HSV-2 replication (Fig 3D–3F). To examine if depletion of macrophages could abrogate HSV-2 induced immunopathology in the absence of any lymphoid cells, we repeated the use of clodronate in NRG mice given α-IFNAR neutralizing Ab. This also resulted in significant abrogation of HSV-2-induced tissue damage (Fig 3G and 3H). These results demonstrate that type I IFNs regulate macrophage function to prevent or minimize tissue damage during HSV-2 infection. PPT PowerPoint slide

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TIFF original image Download: Fig 3. Phagocytic cells are required for HSV-2-induced immunopathology in Ifnar-/- mice. (A) Total number of CD45+F480+ cells in vaginal tissue following infection (n = 5). (B) %CD45+F480+ to total CD45+ cells in vaginal tissue of WT and Ifnar-/- mice at 0 and 3 dpi following HSV-2 infection (n = 5). (C) Schematic of Ifnar-/- mice and NRG mice receiving clodronate or PBS control liposomes. (D) H&E staining of vaginal cross-sections of HSV-2-infected WT and Ifnar-/- mice administered PBS or clodronate liposomes at 3 dpi (n = 5–6). (E) Pathological score of (D). (F) Viral titers of vaginal washes at 1 and 3 dpi of HSV-2-infected Ifnar-/- mice given PBS or clodronate liposomes (n = 4–5). (G) H&E staining of vaginal cross-sections of HSV-2-infected NRG + α-IFNAR Ab mice with PBS or clodronate liposomes at 3 dpi (n = 4–5). (H) Pathology score of (G). 10x scale bar represents 200 μm, 20x scale bar represents 100 μm. Data in (A), (B), (E), (F), (H) and are represented as mean ± SEM. *p < 0.05, and ****p < 0.0001 (A, B, F, two-way ANOVA; E, one-way ANOVA; H, two-tailed t-test). See also S3 Fig. https://doi.org/10.1371/journal.ppat.1010471.g003

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[1] Url: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1010471

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