key: cord-0323847-jo8vg37k authors: Srivastava, Ruchi; Agrawal, Anshu; Vahed, Hawa; BenMohamed, Lbachir title: Age-Related Impairment of Innate and Adaptive Immune Responses Following Intranasal Herpes Simplex Infection date: 2022-01-06 journal: bioRxiv DOI: 10.1101/2022.01.05.475170 sha: 8ec7eb45fd360b0a986102db78cb794f656c19b1 doc_id: 323847 cord_uid: jo8vg37k Immune function declines with age, leading to an increased vulnerability to respiratory viral infections. The mechanisms by which aging negatively impacts the innate and adaptive immune system leading to enhanced susceptibility to infections remain to be fully elucidated. In the present study, we used a mouse model of intranasal infection with herpes simplex virus type 1 (HSV-1), a virus that can enter the lungs through the nasal route causing pneumonia, a serious health concern in the elderly. Following intranasal inoculation of young (6 weeks), adult (36 weeks), and aged (68 weeks) with HSV-1 (KOS strain) we: (i) compared the local and systemic innate and adaptive immune response to infection; and (ii) correlated the level and type of immune response to protection against HSV-1 infection. Compared to young and adult mice, aged mice displayed: (i) increased basal level activation of epithelial cells with a decreased expression of TLR3; (ii) increased activation of dendritic cells with increased expression of MHC-1, MHC-II and CD80/86; and (iii) decreased production of type-I interferons upon stimulation; (iv) a delay in cytokines and chemokines production in the lungs; and (v) an impairment in function (CD107 and IFN-γ production) of HSV-specific CD8+ T cells. These impairment in innate and adaptive immune responses in aged mice following intranasal HSV-1 inoculation was associated with symptomatic herpes infection. The findings suggest an age-related impairment of both innate and adaptive immune responses which may exacerbate herpes infection and disease in the elderly. IMPORTANCE Immune function declines with age, leading to an increased vulnerability of the elderly to respiratory viral infections. The mechanisms by which aging negatively impacts the innate and adaptive immune system leading to enhanced susceptibility to respiratory infections remain to be fully elucidated. The present study showed that, compared to young and adult mice, aged mice displayed increased basal level activation of epithelial cells with a decreased expression of TLR3 increased activation of dendritic cells associated with an impairment of HSV-specific CD8+ T cell responses. These immune dysregulations were associated with to symptomatic herpes infection. The findings suggest an age-related impairment of both innate and adaptive immune responses which may exacerbate herpes infection and disease in the elderly. Herpes simplex virus type 1 (HSV-1) causes a variety of infections that involve 70 mucocutaneous surfaces, the central nervous system and, occasionally, visceral organs such as the 71 lungs (1), (2). Seventy percent of the human population infected with HSV are adults above the age of 72 65.Infection of the respiratory tract with HSV causes herpes simplex pneumonia (HSP). Immune 73 function declines with age, making elderly people more susceptible to pulmonary viral infections. The virus has been reported to be associated with pulmonary disease since 1949 (3). Pulmonary innate immune responses depend upon a highly regulated multicellular network to defend Since the elderly population is increasing worldwide, a better understanding of the changes 89 that the immune system undergoes with the aging process is becoming a key factor in the Briefly, Vero cells were grown in an α-modified Eagle's medium (ThermoFisher Scientific, Waltham, MA) supplemented with 5% fetal bovine serum and 1% penicillin-streptomycin, and L-136 glutamine (ThermoFisher Scientific). For plaque assays, Vero cells were grown to confluence in 24-137 well plates. Nasal wash samples were added to monolayers. Infected monolayers were incubated for 138 1 hour at 37°C and were rocked every 15 minutes for viral absorption. Infected monolayers were 139 overlaid with media containing carboxy-methyl cellulose. Infection was allowed to occur for 72 hours at 37°C. Monolayers were then fixed and stained with crystal violet. Subsequently, the viral plaques 141 were counted under a light microscope. Tissue Harvesting and lung cell isolation: Lungs were harvested (n = 5 per time point per 144 experiment) on days 2 and 6 post-infection. Lungs were exposed by opening the chest cavity and 145 rinsed with cold 1× PBS, through the right heart ventricle. Mice lung tissues were harvested, minced, 146 and digested in 5 mg/ml collagenase for 45 minutes, before filtering through a 70 µm cell strainer. Subsequently, cells were spun down and diluted to 1 × 10 6 viable cells per ml in RPMI media with 148 10% (v/v) FBS. Viability was determined by Trypan blue staining. suggesting that aged AECs were not responding to HSV-1 infection ( Fig. 1C and 1D) . Altogether, these results indicate that AECs from aged mice are impaired in their capacity to sense, respond to 206 infections, and display an increased basal level of epithelial activation. 209 homeostasis: Next, we examined the expression of TLR3 on DCs and macrophages since these 210 cells also get activated via TLRs ( Fig. 2A) . The single cell lung suspension prepared above ( Fig. 1) was also utilized for these experiments. To elucidate the consequences of aging on TLR expression of MHC-I, while no significant change was seen in the adult versus the aged mice (Fig. 3A) . However, 234 lung macrophages MHC class I expression was similar between the young, adult, and aged HSV-1 235 infected mice groups. Despite that lung macrophages from adult mice appeared to up-regulate MHC class I quicker than macrophages from young animals (Fig. 3B) . Similar MHC-I up-regulation patterns 237 on lung lymphoid DCs from young, adult and aged mice were detected (Fig. 3C) . frequencies of CD4 + and CD8 + T cells were induced in the herpes infected group as compared to the mock infected group in all three groups of mice which further declined upon aging (Fig. 7A) . Average 286 frequencies and absolute numbers of CD8 + and CD4 + T cells were detected in the lung of HSV-1 287 infected and mock-infected control group (Fig. 7B) . Then, we compared the frequency of CD8 + T cells week-old mice. As shown in Fig. 8C , significantly high frequencies of functional IFN-γ + CD8 + T cells 305 were detected in HSV-1 infected mice compared to mock infected mice (Fig. 8D) with a significant 306 decline of these cells in the 68-week old mice. These findings indicate that HSV-1 infected 6-and 36- week-old mice induced more functional CD8 + T cells, however, their presence was significantly 308 impacted with the 68-week old mice. sensing viral nucleic acid exposed during viral replication (Fig. 1) . This may be partially responsible age-associated reduced surface expression of TLR3 (Fig. 2) . We have previously reported reduced In this study, we showed that IFN-α response from the lung supernatant was upregulated after Consistent with the delayed production of the cytokines, CD4 + and CD8 + T cells showed delayed 393 infiltration into the lungs of aged animals (Figs. 6A and 6B) . In a primary infection, herpes specific 394 cells might play a more prominent role than non-specifically activated cells. Herpes specific CD8 + T 395 cells were assayed using gB 498-505 immunodominant epitopes. Consistent with our hypothesis gB 498-505 396 specific activated CD8 + T cells were consistently higher in young and adult mice compared to aged 397 mice (Figs. 7C and 7D) . T cell responsiveness to type I IFN, commonly produced in viral and bacterial 398 infections, is known to be pivotal for the generation of adaptive immune responses. Our findings from 399 this report identified a reduced IFN-α response in aged mice that may contribute to reduced T cell 400 responses. The up-regulation of the functional marker CD107 was delayed on CD8 + T cells in aged 401 mice (Fig. 8D) . Previous reports had shown no alterations of CD8 + T cells with age in humans and In this study, we demonstrated that age affects the immune responses to herpes infection. Influenza-induced production of interferon-alpha is defective in geriatric individuals. J Clin Immunol 30:373-83. Herpes simplex viruses Infections with herpes simplex viruses (2) Isolation of herpes virus from a case of atypical pneumonia and 486 erythema multiforme exudativum with studies of four additional cases Virus interference. I. The interferon Toll-like receptors in the pathogenesis of human 490 disease A genital tract peptide epitope vaccine targeting TLR-2 493 efficiently induces local and systemic CD8+ T cells and protects against herpes simplex virus 494 type 2 challenge Targeting the genital tract mucosa with a lipopeptide/recombinant adenovirus prime/boost 497 vaccine induces potent and long-lasting CD8+ T cell immunity against herpes: importance of 498 Quantifying Memory CD8 T Cells Reveals Regionalization of Cutting edge: resident memory CD8 T cells 503 occupy frontline niches in secondary lymphoid organs Impaired interferon 581 signaling in dendritic cells from older donors infected in vitro with West Nile virus IL-22 is essential for lung epithelial repair following influenza infection Directing traffic: IL-17 and IL-22 coordinate pulmonary immune 587 defense IL-22 mediates mucosal host defense against Gram-negative bacterial 591 pneumonia IL-22 reduces the pulmonary injury and lethality of influenza 593 infection (CCR4P.201) Pathological versus 595 protective functions of IL-22 in airway inflammation are regulated by IL-17A The immune response of aged mice to influenza: diminished T-598 cell proliferation, interleukin 2 production and cytotoxicity Requirement of mature dendritic cells 601 for efficient activation of influenza A-specific memory CD8+ T cells Immunity 603 to influenza: the challenges of protecting an aging population Impaired antigen-induced CD8+ T cell clonal expansion in aging is due to defects in antigen 606 presenting cell function Intrinsic versus environmental 608 influences on T-cell responses in aging Alterations in dendritic 610 cell function in aged mice: potential implications for immunotherapy design Murine [corrected] myeloid dendritic cell-dependent toll-like receptor immunity is 614 preserved with aging Figure 1: Decreased TLR3 expression and increased activation of airway epithelial cells 637 (AECs) in aged mice at homeostasis: 6-week, 36-week and 68-week-old mice were infected 638 intranasally with 1 x 10 6 pfu of HSV-1 strain. Mice were euthanized 2 days post infection and a single 639 cell suspension from lungs were obtained after collagenase treatment. The lung cells were stained for 640 epithelial cell markers, TLR-3 and activation markers and then analyzed by FACS. (A) Timeline of 641 infection and immunological analyses Singlets were 643 selected by plotting forward scatter area (FSC-A) vs. forward scatter height (FSC-H). CD45 negative 644 cells were then gated. The epithelial cell population in CD45-gated cells was defined by the EpCAM + 645 cells. (C) Representative histograms and average mean fluorescence intensity (MFI) of TLR-3 646 expression on the surface of lung epithelial cells (D) ICAM expression on epithelial cells (E) MHCI 647 expression on lung epithelial cells of 6-week, 36-week and 68-week-old mice at 2 days post-infection 648 with HSV-1 (solid black) or mock-infection with DMEM Figure 2: The expression of TLR3 on DCs and macrophages in the lung decreases with 652 age at homeostasis. 6-week, 36-week and 68-week-old mice were infected intranasally with 1 x 10 The lung cells were stained for dendritic cell 655 markers and activation markers and then analyzed by FACS. (A) Timeline of infection and 656 immunological analyses. (B) Gating strategy used to characterize lung derived cells. Lymphocytes 657 were identified by a forward scatter (FSC) and side scatter (SSC) gate. Singlets were selected by 658 plotting forward scatter area (FSC-A) vs. forward scatter height (FSC-H). CD45 positive cells were 659 then gated by the expression of CD45. The myeloid cell population in CD45 + gated cells was defined 660 by the CD11b + CD11c + cells. Similarly, CD11b + subset was defined as macrophages and CD11c + 661 cells as lymphoid cell subsets. Representative histograms and average mean fluorescence intensity macrophages (CD11b + CD11c − ) and (E) dendritic cells (CD11c + ) from 6-week, 36-week and 68-week-664 old mice at 2 days post-infection with HSV-1 (solid black) or mock-infection with DMEM Data is representative of two separate experiments The upregulation of MHC-I after herpes infection is impaired with age. 6-week, 668 36-week and 68-week-old mice were infected intranasally with 1 x 10 6 pfu of HSV-1 strain. 2 days 669 post infection mice were euthanized and single cell suspension from lungs were obtained after 670 collagenase treatment. The lung cells were stained for dendritic cell markers and activation markers 671 and then analyzed by FACS. CD45 positive cells were then gated by the expression of CD45. The 672 myeloid cell population in CD45 + CD11b + subset was defined as macrophages and CD11c + cells as lymphoid cell subsets Representative histograms of MHC-I expression mean fluorescence intensity (MFI) on the surface of 675 (A) myeloid cell subset (CD11b + CD11c + ), (B) macrophages (CD11b + CD11c − ) and (C) dendritic cells 676 (CD11c + ) from 6-week, 36 week and 68-week-old mice at 2 days post-infection with HSV-1 (solid 677 black) or mock-infection with DMEM Age-Related Immune Responses to Herpes Infection 6-week, 681 36-week and 68-week-old mice were infected intranasally with 1 x 10 6 pfu of HSV-1 strain. Two days 682 post infection mice were euthanized and single cell suspension from lungs were obtained after 683 collagenase treatment. The lung cells were stained for dendritic cell markers and activation markers 684 and then analyzed by FACS. CD45 positive cells were then gated by the expression of CD45. The 685 myeloid cell population in CD45 + CD11b + subset was defined as macrophages and CD11c + cells as lymphoid cell subsets A) myeloid cell subset (CD11b + CD11c + ), (B) macrophages (CD11b + CD11c − ) and (C) dendritic cells 689 (CD11c + ) from 6-week, 36 week and 68-week-old mice at 2 days post-infection with HSV-1 (solid 690 black) or mock-infection with DMEM Aged mice display reduced secretion of protective cytokines in the lung after 693 Multiplex detection was used to quantitate the levels of inflammatory mediators 694 in the lungs of 6-week, 36-week and 68-week-old mice day 2 post infection. Graphs depict the mean 695 Viral titers and severity of HSV lung infection increases with age. 6-week, 36-698 week and 68-week-old mice were infected intranasally with 1 x 10 6 pfu of HSV-1 strain. Mice were 699 euthanized on day 6 post infection and single cell suspension from lungs were obtained after 700 collagenase treatment. The lung cells were stained for T cell markers and then analyzed by FACS Timeline of infection and immunological analyses. (B) Gating strategy used to characterize lung 702 derived cells. Lymphocytes were identified by a forward scatter (FSC) and side scatter (SSC) gate Singlets were selected by plotting forward scatter area (FSC-A) vs. forward scatter height (FSC-H) C) Nasal 705 swabs and eye swabs were collected from 6-week, 36-week and 68-week-old mice on day 2 and on 706 day 6 for viral titer estimation. HSV-1 DNA copy numbers detected in the nasal swab, lung and eye 707 swab on day 2 and day 6 post infection. (D) Representative nostril images of naïve and infected 6-708 week, 36-week and 68-week-old mice The generation of HSV specific tetramer positive CD8 + T cells is impaired in 711 aged mice. 6-week, 36-week and 68-week-old mice were infected intranasally with 1 x 10 The lung cells were stained for T cell markers and then 714 analyzed by FACS. (A) Representative FACS plots of the frequencies of CD4 + and CD8 + T cells 715 detected in the lung of HSV-1 infected and mock-infected control group (B) Average frequencies and 716 absolute numbers of CD8 + and CD4 + T cells detected in the lung of HSV-1 infected and mock-infected 717 control group. (C) Representative FACS plots of the frequencies of gB 498-505 specific CD8 + T cells 718 detected in the lung of HSV-1 infected and mock-infected control group (D) Average frequencies and 719 absolute numbers of gB 498-505 specific CD8 + T cells detected in the lung of HSV-1 The cytotoxic and functional activity of CD8 + T cells is reduced with age in HSV 723 infected mice. 6-week, 36-week and 68-week-old mice were infected intranasally with 1 x 10 Seven days post infection mice were euthanized and single cell suspension from lungs 725 were obtained after collagenase treatment. (A) Representative FACS plots of the frequencies of 726 CD107 + CD8 + T cells detected in the lung of HSV-1 infected and mock-infected control group (B) Average frequencies and absolute numbers of CD107 + CD8 + T cells detected in the lung of HSV-1 728 infected and mock-infected control group γ + CD8 + T cells detected in the lung of HSV-1 infected and mock-infected control group (D) Average 730 frequencies and absolute numbers of IFN-γ + CD8 + T cells detected in the lung of HSV-1 infected and 731 mock-infected control group. Data is representative of two separate experiments.