Sections 5 to 15 m thick were then prepared from OCT-embedded samples and fixed in acetone for 10 minutes at 4C

Sections 5 to 15 m thick were then prepared from OCT-embedded samples and fixed in acetone for 10 minutes at 4C. of the 3 pathogenic human coronaviruses because virtually no virus-specific antibodies or follicular helper CD4+ T (Tfh) cells were produced. Using MERS-CoVCinfected mice, we found that these effects did not result from T or B cell intrinsic factors. Rather, they resulted from enhanced, and ultimately, pathogenic rDC activation, as manifested most prominently by enhanced IL-1 expression. Wild-type rDC transfer to mice in conjunction with partial IL-1 blockade reversed this defect and resulted in increased virus-specific antibody and Tfh responses. Together, these results indicate that PLA2G2D has an unexpected role in Acarbose the lungs, serving as an important modulator of rDC activation, with protective and pathogenic effects in respiratory coronavirus infections and immunization, respectively. mice as a consequence of enhanced respiratory dendritic cell (rDC) migration from the lungs to draining lymph nodes and subsequent augmented virus-specific T cell responses (13). Since vaccination efficacy is lower in aged compared with young mice, we reasoned that the absence of PLA2G2D would also enhance memory T cell and antibody responses after intranasal (i.n.) immunization with sublethal virus infection or with other immunogens, thereby increasing vaccine efficacy. To assess this possibility, we immunized and infected mice with MERS-CoV, SARS-CoV, or SARS-CoV-2. Most of the studies described below study MERS-CoVCinfected mice. Mice were immunized with a sublethal dose of MERS-CoV (sublethal infection) or with a replication-incompetent alphavirus replicon particle expressing the spike protein or nucleocapsid of MERS-CoV (VRP-MERS-S, VRP-MERS-N). Mice are normally resistant to infection with MERS-CoV, but are rendered susceptible to the virus if the human receptor human dipeptidyl peptidase-4 (hDPP4) is present. For this purpose, we generated mice knocked-in for and then further adapted the virus to mice by serial passage through these mice (16). We then produced mice lacking expression (mice) by crossing the mice with mice. To study SARS, we directly infected mice with mouse-adapted SARS-CoV, as previously described Acarbose (13). Mice are naturally resistant to SARS-CoV-2, but become susceptible if the human receptor (human angiotensin converting enzyme-2 [hACE2]) is provided using an adenovirus encoding hACE2 (Ad5-hACE2; refs. 17, 18). Use of Ad5-hACE2 transduction allowed for infection of and mice with human strains of SARS-CoV-2. Here, in contrast to results observed in mice with acute respiratory CoV infection, we found that i.n. immunization of middle-aged (5C8 months old) but not young (8 weeks old) mice resulted in no improvement in outcomes after MERS-CoV challenge because virtually no virus-specific antibody was produced. Similar results were found in mice infected with SARS-CoV or SARS-CoV-2. Antibody deficiency Acarbose resulted from a nearly complete absence of a follicular helper CD4+ T cell (Tfh) response. As in the acute infection, activation of rDCs was enhanced in mice, but in this case, it resulted in poor Tfh cell and virus-specific antibody production. Results PLA2G2D deficiency impairs protection in middle-aged but not young mice after MERS-CoV immunization and challenge. To address the role of PLA2G2D in memory immune responses, we immunized middle-aged (5C6 month old) and mice with a sublethal dose of virus (Figure 1, ACC) or VRP-MERS-S (Figure 1, DCI) and then challenged them with a lethal dose of MERS-CoV. Prior to analyzing these mice, we found that acute MERS-CoV infection of naive middle-aged mice resulted in more rapid kinetics of virus clearance (Figure 1, B and E), decreased pathological damage (Supplemental Figure 1, A and B; supplemental material available online with this article;, and diminished morbidity and mortality (Figure 1, C and F) compared with infected mice, consistent with studies of SARS-CoV (13). In contrast, while immunization with a sublethal dose of MERS-CoV protected middle-aged mice from weight loss and death and enhanced the kinetics of virus clearance, it did not increase survival of middle-aged mice (Figure 1, B and C). Open in a separate window Figure 1 PLA2G2D deficiency impairs immunization-induced protection in middle-aged mice challenged with MERS-CoV.(A) Protocol for MERS-CoV sublethal infection and challenge. Six-month-old or mice were immunized with a sublethal dose (100 pfu) of MERS-CoV or PBS i.n. on day 0, followed by i.n. infection with a lethal dose (750 pfu) of MERS-CoV on day 28. Virus titers in lungs at 7 dpi (6) (B) and survival (8C10/group) (C) of GGT1 naive and immunized 6-month-old or mice are shown. (D) Protocol for VRP-MERS-S immunization and MERS-CoV infection. 8-week-old (young) or 5-month-old (middle-aged) or mice were treated with VRP-MERS-S or PBS i.n. on day 0 and day 28, followed by a lethal dose (250 pfu for young mice and 750 pfu for middle-aged mice) of MERS-CoV i.n. on day 70. (E) Virus titers in lungs of middle-aged or mice at 7 dpi are shown, 6/group. (F) Survival of infected middle-aged mice, 8 to 10/group. (G) Survival of infected young (8-week-old) mice, 8 to 10/group. (H) Protocol for i.p. VRP immunization. Five-month-old or mice were immunized with VRP-MERS-S or PBS i.n. on.