key: cord-0283113-z47n51h9
authors: Chambers, Emma S; Vukmanovic-Stejic, Milica; Shih, Barbara B; Trahair, Hugh; Subramanian, Priya; Devine, Oliver P; Glanville, James; Gilroy, Derek; Rustin, Malcom; Freeman, Tom C; Mabbot, Neil A; Akbar, Arne N
title: Monocyte-derived Prostaglandin E2 inhibits antigen-specific cutaneous immunity during ageing
date: 2020-04-03
journal: bioRxiv
DOI: 10.1101/2020.04.02.020081
sha: 6d037195823c7cdd4048e822a0b80dd3d9ff109a
doc_id: 283113
cord_uid: z47n51h9

Ageing results in a decline in immune function. We showed previously that healthy older humans (>65 years old) have reduced antigen-specific cutaneous immunity to varicella zoster virus (VZV) antigen challenge. This was associated with p38 MAP kinase driven inflammation that was induced by mild tissue injury caused by the injection of the antigen itself. Here we show that non-specific injury induced by injection of air or saline into the skin of older adults recruits CCR2+CD14+ monocytes by CCL2 produced by senescent fibroblasts. These monocytes reduced TRM proliferation via secretion of prostaglandin E2 (PGE2). Pre-treatment with a p38-MAPK inhibitor (Losmapimod) in older adults in vivo significantly decreased CCL2 expression, recruitment of monocyte into the skin, COX2 expression and PGE2 production. This enhanced the VZV response in the skin. Therefore, local inflammation arising from interaction between senescent cells and monocytes leads to immune decline in the skin during ageing, a process that can be reversed. Summary Inflammation resulting from tissue injury blocks antigen-specific cutaneous immunity during ageing. Monocytes recruited to the skin inhibit TRM function through COX2-derived prostaglandin E2 production. Blocking inflammation and resulting prostaglandin E2 production with a p38-MAP kinase inhibitor significantly enhances cutaneous antigen-specific responses.

For experiments involving co-culture of monocytes with skin TRM cells, two suction blisters 226 were formed over uninjected skin, as described previously (Akbar et al., 2013) . Cells were 227 collected from the blister the following day and incubated on plastic for 3 hours, to remove 228 mononuclear phagocytes. Cell suspensions (predominantly CD4 + T cells) were removed and 229 labelled with CellTrace Violet and co-cultured with unstimulated or LPS stimulated 230 monocytes as described above. Life Technologies) and boiled for 5 min at 95 °C. Samples were separated by protein 243 electrophoresis at 120 V for 2 h using 10% Bis-Tris pre-cast gels (NuPage) and transferred 244 overnight at 4°C onto Hybond-P PVDF membranes (GE Healthcare). After blocking in ECL 245 blocking agent (GE Healthcare), membranes were probed with primary antibodies overnight 246 at 4°C. Primary antibodies used were COX2 (ab15191; Abcam), phospho-p38 MAPK 247 (T180/Y182; 9211), and p38 MAP Kinase (9212), GAPDH (2118; Cell Signalling). The 248 membrane was washed and incubated with HRP-conjugated secondary antibodies (GE 249 Healthcare, 1:4000) for 1 h at room temperature. Antibodies were detected using the ECL 250 detection kit (GE Healthcare). Prior to re-probing with different antibodies, membranes were 251 stripped at room temperature with agitation using Restore stripping buffer (Thermo 252 Scientific). Protein bands were quantified using ImageJ software. The integrated density of 253 each band was normalised to GAPDH using the gel analysis function of ImageJ. 254 255 Dermal Fibroblast isolation and culture: 256

Dermal fibroblasts were isolated and cultured as described previously (Pereira et al., 2019) . 257 senescence was induced in primary human dermal fibroblasts by exposure to X-ray radiation 258 at a total dose of 10Gy at a rate of 5Gy/min. Irradiated cells were cultured for a further 10-28 259 days to allow for senescence to develop. Senescence was confirmed by staining for SAβ-260 galactosidase (Cell signalling, London U.K. 

characteristics can be found in Table 1 . Although there were more females in our older adult 280 group, there was no difference in response between male and female older adults 281 (Supplementary Figure 1A) . Volunteers were injected intradermally with VZV skin test 282 antigen in one arm (for the clinical score) and with saline (0.9% NaCl) in the other arm as a 283 control. 5mm biopsies were collected from the injection sites 6 hours post-injection and 284 compared to biopsies of normal (unmanipulated) skin ( Figure 1A ). We showed previously 285 that mononuclear phagocyte numbers were increased at the site of saline injection in old but 286 not young subjects and that this accumulation was negatively correlated with the magnitude 287 of antigen-specific responses induced in the contralateral arm of the same individual 288 To enumerate the changes in monocyte numbers following saline injection we assessed 293 paired samples and expressed data as delta change from normal skin. This was to mitigate 294 against inter-personal variation in the magnitude of responses observed (Supplementary 295 Figure 2A ). CD14 is expressed by the majority of peripheral blood monocytes (classical and 296 intermediate). There was a significant increase in CD14 + cells in saline injected skin at 6 297 hours post-injection ( Figure 1B and 1C) . We next investigated the expression of CCR2, a 298 chemokine receptor for CCL2, which is highly expressed by CD14 + monocytes (Patel et al., 299 2017; Ziegler-Heitbrock et al., 2010). There was a significant increase in number of 300 CD14 + CCR2 + cells in saline injected skin as compared to normal skin in old but not young 301 subjects ( Figure 1C ). This response to saline was transient as by 24 hours post-injection 302 there is was no significant difference in frequency of monocytes between normal and saline-303 injected skin (Supplementary Figure 2C) . Multiparametric flow cytometry analysis of normal 304 and saline biopsies, using our previously published panel to identify mononuclear 305 phagocytes (Vukmanovic-Stejic et al., 2018), confirmed that there was a significant increase 306 in CD14 + and CCR2 + CD14 + cells in old saline-injected skin ( Figure 1D ). 307

To investigate whether this inflammatory response after saline injection in older humans was 308 due to sodium chloride itself or a response to needle injury, we performed intradermal 309 injections with the same volume of air instead of saline (20μl). Air injection also induced a 310 significant increase in the number of monocytes at the site of air challenge in old but not 311 young subjects compared to normal skin in both groups (Supplementary Figure 2B) . The 312 accumulation of monocytes was also observed at six hours at the site of VZV injection in old 313 but not young adults ( Figure 1E ). Taken together these data suggest that old subjects have 314 a heightened inflammatory response to mild skin injury that is not observed in young 315

We previously showed that >85% of older individuals have a low VZV response, clinical 317 score between 0-3, while >85% of young individuals have a score of between 3-10 (Agius et 318 al., 2009; Vukmanovic-Stejic et al., 2015). However, a small proportion of older people 319 (~15%) show good cutaneous VZV responsiveness (score 3-10), a group we designated as 320 'old responders'. To assess whether the CD14 + monocyte infiltration was a characteristic of 321 all aged individuals or only those with reduced VZV-specific immunity, we investigated the 322 infiltration of these cells after saline in old VZV responders (red circles) and low responders 323 (filled circles). Old responders to VZV exhibited a similar low extent of CD14 + monocyte 324 infiltration to younger adults (black squares). However, the majority of older donors with a 325 low clinical score (filled circles) had elevated infiltration of these cells. When all the data was 326 combined there was a significant negative correlation between the fold change in CD14 + 327 monocytes in response to saline challenge and the VZV clinical score (R=-0.38 and p=0.04; 328 Figure 1E ). Collectively this indicates that the early recruitment of monocytes to the site of 329 skin challenge in old people was associated with decreased antigen-specific immunity. 330 331 Senescent stromal cells contribute to recruitment of monocytes in the old. 332

We hypothesised that the increase in CD14 + cells in the skin was due to recruitment of 333 circulating monocytes, since CCR2 + expression supported the possibility of recruitment in 334 response to CCL2. The alternative explanation that proliferation of local tissue-resident 335 macrophages (identified as CD163 + ) was not observed (data not shown). This is in line with 336 murine studies showing dermal macrophages are constantly replenished from the monocyte 337 pool (Tamoutounour et al., 2013) . We first investigated the activation status of the dermal 338 endothelium to determine whether it was permissive for monocyte recruitment. CD62E, also 339 known as E-selectin, was used as a marker of activated endothelium as it binds to sialylated 340 carbohydrates expressed on leukocytes and facilitates their extravasation into the tissue. 341

Saline-injected skin of old but not young adults exhibited increased CD62E expression on 342 the CD31 + endothelium when compared to normal skin (Figure 2A and B). Furthermore, 343 transcriptional analysis of skin biopsies indicated that there was increased expression of a 344 range of genes encoding monocyte chemoattractants including CCL2, CXCL10, CXCL2 and 345 CXCL1 in old but not young saline-injected skin as compared to normal skin ( Figure 2C ).

The increase of CCL2 expression (one of the main monocyte chemoattractants) was as a marker of fibroblasts, we found that there was a significant increase in the number of 351 fibroblasts expressing CCL2 in the skin of old subjects after saline injection as compared to 352 normal un-injected skin from old donors ( Figure 2E volunteers. Immunofluorescence staining was performed on normal skin to determine 408 expression of EP4 on CD4 + TRM (as identified by co-expressing CD69; Figure 4A ). The 409 majority of CD4 + TRM expressed EP4 (81.2%), indicating that they have the capacity to 410 respond to PGE2 produced by monocytes. We next assessed the co-localisation of CD14 + 411 monocytes and CD4 + TRM cells in normal and saline injected skin from old subjects. In normal 412 skin, CD14 + monocytes were on average 15.86μm away from CD4 + TRM, however after 413 saline injection the monocytes were significantly closer to the CD4 + TRM (9.47μm; Figure 4B  414 and C). This increased proximity was likely due to increase in the number of monocytes in 415 the skin after saline injection. To confirm that monocytes have the ability to suppress skin 416 CD4 + TRM proliferation, CD4 + TRM were isolated from the skin using suction blister technology 417 as described previously ( Figure 4D ) (Akbar et al., 2013) . Skin CD4 + TRM cells were pre-418 labelled with CellTrace Violet and stimulated with CD3 and IL-2 in the presence of either 419 stimulated or unstimulated monocytes for four days. We observed a significant reduction in 420 CD4 + TRM cell proliferation in the presence of stimulated when compared to unstimulated 421 monocytes ( Figure 4E Figure 4B) . Furthermore, after Losmapimod there was a 445 significant reduction in the number of CCL2 + cells and the number of CCL2 + senescent 446 fibroblasts as defined as being p16 + FSP-1 + (Figures 5D, 5E) . We confirmed experimentally 447 that the treatment of senescent dermal fibroblasts with Losmapimod in vitro significantly 448 reduced their CCL2 production, suggesting Losmapimod could have a direct effect on 449 senescent fibroblast in vivo (Supplementary Figure 4C) . As a result of the reduced CCL2 450 there was significantly reduced monocyte infiltration in saline injected skin Post-Losmapimod 451 ( Figure 5F ). In addition, when VZV injected skin (6 hour post-injection) was assessed there 452 was a significant reduction in the number of monocytes after Losmapimod treatment.

This data indicates that p38-MAPK inhibition significantly reduces CCL2 production from antigen challenge in the skin ( Figure 5G) . 456 457 p38 MAP kinase inhibition significantly inhibits PGE2 that restores T cell proliferation.

in vivo. To test this, we performed Western blot analysis of LPS activated monocytes in the 461 presence or absence of Losmapimod. Losmapimod significantly decreased the LPS induced 462 expression of phopho-p38 MAPK and COX2 in vitro (Figures 6A and B ). In addition, PGE2 463 production was also significantly decreased after Losmapimod treatment ( Figure 6C ). 464

Furthermore, the addition of Losmapimod to co-cultures of CD4 + T cells that were activated 465 in the presence of LPS treated monocytes ( Figure 6D ) enhanced their proliferation to a 466 similar extent to that observed after direct COX2 or EP4 inhibition ( Figure 3G and H). In line 467 with these observations, the increase in CD14 + COX2 + cells after saline injection was also 468 inhibited after post-Losmapimod treatment in vivo ( Figure 6E ). These data collectively 469 suggest that in vivo Losmapimod can restore CD4 + T cell responses in part through the 470 inhibition of COX2 expression. 471 472

We assessed the clinical score of 42 old subjects after VZV antigen challenge in the skin 475 both before and after Losmapimod treatment. We confirmed and extended our previous 476 observations that treatment with Losmapimod significantly increased VZV clinical score 477 (n=42; Figure 7A ). No difference was observed between male and female donors in their 478 response to Losmapimod treatment (Supplementary Figure 1B) . Losmapimod treatment also 479 significantly decreased serum CRP in all individuals, irrespective of whether or not they 480 increased their skin response to VZV challenge ( Figure 7B) . 481

We showed previously that there is a significant association between the clinical score after 482 symptoms (Minfeng Liao and Li, 2020). Since the majority of the patients with most severe symptoms are older (Stephanie Bialek, 2020), it will be important to determine the 564 mechanism by which these "inflammatory" monocytes are recruited into the lung. It is 565 possible that once inflammatory monocytes are recruited into the lungs of these patients 566 they may inhibit the function of virus-specific T cells, by the mechanism that we have 567 described, thus preventing appropriate anti-viral immunity (Stephanie Bialek, 2020) . 568

Relevant to our current observations, it has been shown that senescent cells increase in the 569 lungs with age-dependent pathology ( 

A, Heatmap showing the expression of genes associated with inhibitory mechanisms in normal and saline injected of old and young donors B, representative staining of DAPI (white), COX2 (red) and CD14 (blue) in normal and saline injected skin and C, cumulative data of total COX2 expressing cells and D, number of CD14 + COX2 + cells in normal and saline injected skin. E, Monocytes were negatively isolated from the peripheral blood and cultured with and without LPS for 3 hours then subsequently co-cultured with autologous T cells (pre-labelled with CellTrace Violet) that were activated with plate-bound CD3 and IL-2, proliferation was assessed at day 4. A representative flow plot of CellTrace violet dilution in CD4 + T cells co-cultured with unstimulated and stimulated monocytes is shown in E and F, cumulative data showing percent of cells divided. A similar co-culture experiment was performed in the presence or absence of the COX2 inhibitor NS-398 or G, the EP4 receptor inhibitor MF498 H. In C,D and F, analysed with a paired t-test. G and H analysed with Wilcoxon matched-pairs signed rank test * = p<0.05; *** = p<0.001 Figure 4 : PGE2 production by monocytes inhibits skin CD4 + TRM cell proliferation. A, representative staining of CD4 (green), CD69 (red) and EP4 (blue) in normal skin, white arrows indicate CD4 + TRM cells which co-express EP4. B, representative staining showing co-localization of CD4 + TRM cells and CD14 + monocytes (blue) and C, cumulative data showing distance of CD14 + monocytes from TRM in normal and saline injected skin from older adults. Monocytes were negatively isolated from the peripheral blood and cultured with and without LPS for 3 hours then subsequently co-cultured with skin TRM cells (prelabelled with CellTrace Violet), which were collected from suction blisters (representative image in D) then activated with plate-bound CD3 and IL-2 and proliferation was assessed at day 4. E, representative flow plot of CellTrace violet dilution in TRM cells co-cultured with unstimulated (red line) or stimulated (blue line) monocytes. F, Cumulative data on CD4 + TRM proliferation in the presence of stimulated or unstimulated monocytes, assessed at day 4. C, and F, were analysed with a paired t-test. * = p<0.05 Figure 5 : Reduced inflammatory monocyte recruitment to the skin by Losmapimod pretreatment A, Losmapimod clinical study diagram. Black arrows indicate study visits, red arrows indicate blood samples collected and blue arrows indicate skin biopsies taken. B, Colour coding of the clinical response of each individual after VZV challenge both before and after Losmapimod pre-treatment (pink/brown). Further colour coding was introduced to identify the extent of improvement where dark green identifies the biggest improvement and white shows no improvement in the skin. C, This coding was used in conjunction with the transcriptomic signatures in normal and saline injected skin before and after Losmapimod treatment. The top 30 genes upregulated in saline injected skin as compared to normal skin before pre-Losmapimod. D, Fold change of CCL2 + cells and E, FSP1 + CCL2 + cells in saline injected pre-and post-Losmapimod treatment. F, fold change CD14 + cell in the skin in response to saline injection and G, number of CD14 + monocytes in VZV injected skin (6 hours after injection) pre-and post-Losmapimod treatment. Significance in D-G assessed using a paired t-test. * = p<0.05

Monocytes were negatively isolated from the peripheral blood and cultured with and without LPS and in the presence or absence of Losmapimod for 3 hours, pellets were collected and western blot performed A, representative blot of phospho-p38 MAP Kinase (p-p38), total p38 MAP Kinase (p38), COX2 and GAPDH. B, cumulative data of COX2 expression relative to GAPDH (n=3) and C, Prostaglandin E2 (PGE2) production. Monocytes were negatively isolated from the peripheral blood and stimulated with LPS (blue) and without LPS (red) or with LPS in the presence of Losmapimod (white) for 3 hours then subsequently co-cultured with autologous T cells (prelabelled with CellTrace Violet) in the presence of plate-bound CD3 and IL-2 proliferation was assessed at day 4. D, cumulative data showing percent of cells divided. E, fold change of infiltration of CD14 + COX2 + cells between normal and saline injected skin before and after Losmapimod treatment. B, and C, analysed by a one-way ANOVA with Tukey's multiple comparison test and D, and E, analysed with a Wilcoxon matched-pairs signed rank test * = p<0.05

A, VZV clinical score in all donors (n=42) and B, serum CRP concentrations pre-and post-Losmapimod treatment. C, representative images of CD4 (red) and Ki67 (green) staining at day 7 days after VZV challenge with and without Losmapimod pre-treatment. D, cumulative data of CD4 + staining and E, CD4 + Ki67 + staining at different times after VZV challenge, pre and post Losmapimod pretreatment. F, representative image of CD8 (red) and Ki67 (green) staining at day 7 after VZV challenge and G, cumulative data of CD8 + staining and H, CD8 + Ki67 + staining at different times after over the time course pre and post Losmapimod pre-treatment. D, E, G, and H analysed with a paired t-test. * = p<0.05

In older adults the mild damage induced by injection (of air, saline or VZV) results in production of CCL2 from senescent fibroblasts and recruitment of monocytes into the skin 6 hours post injection. The recruited monocytes inhibit TRM proliferation via upregulation of COX2 and subsequent production of the lipid mediator Prostaglandin E2 (PGE2). The treatment of older adults with the p38 MAPK inhibitor, Losmapimod, results in reduced CCL2 production from the senescent fibroblasts and reduced monocyte infiltration. In addition, p38 MAPK is upstream of COX2 signalling, therefore the treatment with Losmapimod also inhibits COX2 upregulation and reduced PGE2 production. This significantly increases the T cell response to VZV in the skin of older subjects

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