key: cord-0427795-di6m9gxa
authors: Guo, Kai; Yombo, Dan J.K; Xu, Jintao; Wang, Zhihan; Schmit, Taylor; Tripathi, Jitendra; Hur, Junguk; Sun, Jie; Olszewski, Michal A.; Khan, Nadeem
title: The chemokine receptor CXCR3 promotes CD8+ T cell-dependent lung pathology during influenza pathogenesis
date: 2022-02-14
journal: bioRxiv
DOI: 10.1101/2022.02.14.480379
sha: 3a678e150e568ebb46e85a2ae34deed7372c2236
doc_id: 427795
cord_uid: di6m9gxa

While the role of CD8+ T cells in influenza clearance is established, their contribution to pathological lung injury is increasingly appreciated. To explore if protective versus pathological functions can be linked to CD8+ T cell subpopulations, we dissected their responses in influenza-infected murine lungs. Our single-cell RNASeq (scRNAseq) analysis revealed significant diversity in CD8+ T cell subpopulations during peak viral load vs. infection-resolved state. While enrichment of Cxcr3hi CD8+ T effector (Teff) subset was associated with a more robust cytotoxic response, both CD8+ Teff and CD8+ T central memory (TCM) exhibited equally potent effector potential. The scRNAseq analysis identified unique regulons regulating the cytotoxic response in CD8+ T cells. The neutralization of CXCR3 mitigated lung injury without affecting viral clearance. IFN-γ was dispensable to regulate the cytotoxic response of Cxcr3hi CD8+ T cells. Collectively, our data imply that CXCR3 interception could have a therapeutic effect in preventing influenza-linked lung injury. TEASER The CXCR3 expressing CD8+ T cell subset causes severe lung pathology and exacerbates disease severity without affecting viral clearance during influenza infection

IFN-γ was dispensable to regulate the cytotoxic response of Cxcr3 hi CD8 + T cells. Collectively, 48 our data imply that CXCR3 interception could have a therapeutic effect in preventing influenza-49 linked lung injury. (receptors) signaling between Cxcr3 hi and Cxcr3 low clusters (mock, 7 and 14 dpi), we combined 241 all identified signaling pathways from different datasets. We subsequently compared them in 242 parallel, which allowed us to identify ligand-receptor pairing that exhibited different signaling 243 patterns (Fig. 3 , B and C). Compared with mock, the majority of pathways such as CCL, CXCL, 244 MIF, IFN-I, PARs, ALCAM, CD39, CD6, CD86, CLEC, PD−L1, PDL2, and THY1 were found 245 to be active in Cxcr3 hi cellular clusters at 7 dpi. Among the highly active pathways at 7 dpi, MIF, 246 IFN-I, ALCAM, CD39, CD6, CLEC, and THY1 pathways were significantly downregulated by 247 14 dpi, except for cytotoxicity-triggering receptor NKG2D, which was upregulated in cluster E6 248 at day 14 pi (Fig. 3 , B and C). In contrast, Cxcr3 low clusters exhibited enhanced IL-2 signaling 249 (compared to Cxcr3 hi clusters) at 7 and 14 dpi (Fig. 3 , B and C). Thus, Cxcr3 hi CD8 + T cells display 250 unique molecular pathways associated with cytotoxic response in the lungs during influenza 251 infection. 252 253 Because cytotoxic CD8 + T cell response is regulated by a coordinated function of several 254 transcriptional factors (28, 29), we examined the differences in transcriptional factors and their 255 gene modules, also known as regulons, of Cxcr3 hi vs. Cxcr3 low CD8 + T cells using the single-cell 256 regulatory network inference and clustering (SCENIC) software. A total of 227 regulons with 257 9,619 genes were identified across Cxcr3 clusters, which were further binarized and matched with 258 cell types (Fig. 3D) . Several important regulons, including IFN regulated Tbx21, Nfatc2, and Batf, 259

were uniquely activated in Cxcr3 hi clusters. Additionally, Runx3, Runx2, and Stat3 regulons were 260 highly activated in Cxcr3 hi clusters (Fig. 3E) , and significant downregulation of these regulons 261 was observed in Cxcr3 low cells (Fig. 3E) . Several regulons such as Irf8, Irf7, Stat1, Mafb, Irf5, and Spi1 were commonly activated at 7 dpi and were turned off at 14 dpi in both Cxcr3 hi and Cxcr3 low 263 cells (Fig. 3, D and E) . Notably, consistent with the above findings of chemokine and IFN signaling, 264 cluster E6 represented a majority of activated regulons in Cxcr3 hi clusters, suggesting that cluster 265 E6 is a major contributor of inflammatory response in Cxcr3 hi CD8 + T cells (Fig. 3, D and E) . 266

Overall, these data identified several key regulons implicated in differential regulation of host 267 response in Cxcr3 hi and Cxcr3 low clusters, further supporting that these cells are the major drivers 268 of inflammation and cytotoxicity during influenza infection. we determined the cellular communications for CXCL signaling in Cxcr3 hi and Cxcr3 low CD8 + T 274 cells. Our data show that compared to mock, clusters N0, N11, and N12 from Cxcr3 low and clusters 275 E1 and E4 from Cxcr3 hi cells were more profoundly associated with the expression of CXCL 276 chemokine ligands at 7 dpi (Fig. 3F) . However, E1, E4, and E6 were the main recipient clusters in 277

Cxcr3 hi cells, while N0 was the only recipient cluster in Cxcr3 low cells at 7 dpi (Fig. 3F ). The cluster 278 E6 was the only expressor of CXCL chemokines at day 14 pi, and the clusters E1 and E4 acted as 279 the recipient cells of Cxcr3 hi clusters at 14 dpi (Fig. 3F) . We did not detect the chemokine signaling 280 in Cxcr3 low cells at 14 dpi. The Cxcr3-Cxcl10 and Cxcr6-Cxcl16 ligand-receptor pairs were 281 predominantly expressed at 7 dpi, suggesting that both pathways were involved in the early 282 recruitment of CD6 T cells in the lungs. In contrast, the Cxcl10-Cxcr3 ligand-receptor pair was the 283 only contributor to the CXCL communication pathway at 14 dpi (Fig. 3F, bottom & right) , 284

suggesting that the late-phase CD8 + T cell recruitment was relying solely on the CXCR3 pathway.

We further examined the expression of CXCR3 ligand, Cxcl9, and Cxcl10 in CD8 + T cell clusters. 286

While Cxcl10 expression was detected in each cluster (Cxcr3 hi and Cxcr3 low ), the expression of 287

Cxcl9 remained undetectable ( fig. S3A ), suggesting the CD8 + T cell-independent association of 288

Cxcl9 expression in our model. Next, we analyzed the ligand-receptor interactions of type-I (IFN-289 I) and type-II (IFN-II) interferons. While the clusters from both Cxcr3 low (N5, N7, N11, N14) and 290

Cxcr3 hi (E1, E4, and E6) expressed IFN-I, the cluster E6 was the only cluster recipient of IFN-I 291 signaling (Fig. 3F) . We did not detect IFN-II receptor signaling among Cxcr3 hi or Cxcr3 low cell 292 clusters in mock or IAV infected 7 and 14 dpi time points ( fig.S3B ). Thus, CXCR3/CXCL10 axis 293 is the main pathway for both early and late CD8 + T-cell recruitment in the influenza-infected mice, 294 and among Cxcr3 hi cells, cluster E6 appears to be the main driver of IFN-I dependent inflammatory 295 response in Cxcr3 hi CD8 + T cells. 296

The antibody blockade of CXCR3 mitigates influenza lung injury and disease severity 298 without affecting viral clearance 299

Since CXCR3 + CD8 + T cells showed all the attributes of cells specialized in the most robust 300 cytotoxic response, we postulated the CXCR3 + CD8 + T cells contributed to influenza lung injury. 301

We performed antibody-mediated neutralization of CXCR3 in influenza-infected mice (Fig. 4A ) 302 and examined multiple parameters associated with lung inflammation, injury, and disease severity. 303

The antibody-mediated neutralization of CXCR3 led to an approximately 70% reduction of overall 304 CD8 + T cells in influenza-infected lungs at 7 dpi (Fig. 4 , B and C). We did not observe a 305 quantitative increase in the frequency of CD4 + T cells at 7 dpi, suggesting that CD8 + T cells 306 acquired an early effector and cytolytic phenotype (Fig. 4 , D and E) as early as 7 dpi. Moreover, 307 CXCR3 blockade resulted in a significantly greater loss of CD8 + T cells than CD4 + T cells even 308 at a late time point, 14 dpi. (Fig. 4E ). These findings are consistent with our scRNAseq and flow 309 cytometry data that CXCR3 is primarily expressed by CD8 + T cells in our model. The depletion 310 of CXCR3 resulted in the reduced level of CCL2 chemokine and consequently the reduced CCR2 311 monocytes (Fig. 4, F and G) . The administration of anti-CXCR3 antibody also resulted in 312 significantly reduced levels of CD8 + T cell-specific effector (i.e., IFN-g) and cytolytic molecules, 313 (i.e., perforin, granzyme-B) (Fig. 4 , H to J). Notably, while CXCR3 antibody blockade resulted in 314 a significant loss of CD8 + T cell cytotoxic response, it did not abolish the CD8 + T effector and 315 cytotoxic response altogether. We further measured the levels of CXCR3 cognate binding 316 chemokines CXCL9 and CXL10, as well as cytokines in the lung homogenates of mock-and 317 influenza-infected mice at 7 dpi. We found that CXCR3 antibody blockade resulted in reduced 318 levels of CXCL9 and IL-10 ( Fig. 4 , K to L). The lower IL10 level is indicative of reduced lung 319 inflammation in mice with CXCR3 antibody blockade. Overall, these data demonstrated that 320 CXCR3 blockade dampened the expression of inflammatory and cytolytic molecules in influenza-321 infected lungs. To corroborate our CXCR3 antibody-based neutralization approach, we compared 322 CD8 + T cell cytotoxic response in influenza-infected WT with those of CXCR3 deficient (CXCR3 - We further hypothesized that CXCR3 depletion would improve/reduce inflammation and 330 pathology while sustaining antiviral responses through the function of CXCR3 -CD8 + T cells. We compared multiple parameters of influenza disease severity, i.e., weight loss, inflammation, and 332 acute lung damage, in influenza-infected mice treated with or without anti-CXCR3 antibody. 333

Compared to influenza-infected control mice (WT-PR8), mice treated with CXCR3 antibody (WT-334 PR8-αCXCR3) exhibited significantly reduced lung injury, evidenced by a reduced level of LDH 335 in BAL (Fig. 5A ) and reduced inflammation shown in and H&E tissue pathology at 7 and 14 dpi 336 5F ). While LDH and albumin levels peaked at 7 dpi and significantly reduced by 14 dpi, the 342 H&E tissue-pathology assessments showed a non-resolving lung injury and vascular damage in 343 influenza-infected mice even at 14 dpi. Consistent with reduced lung inflammation and injury, 344 CXCR3-neutralized mice also exhibited a significantly reduced weight loss (Fig. 5H ). These 345 findings were subsequently validated in CXCR3 -/mice showing similarly reduced lung 346 inflammation and pathology evident on histological sections and through the reduced levels of 347 LDH and albumin in the BAL compared to the WT mice ( fig. S5 , A to C). These data demonstrated 348 that the CXCR3 pathway is an important driver of lung injury during the peak viral load (7 dpi) 349 and its disruption facilitates the expedited resolution of lung injury following the viral clearance. 350

Since abolished CXCR3 signaling had such a profound impact on limiting lung pathology, we 351 assessed its role in viral clearance to explore its usefulness as a therapeutic target. Both anti- We further addressed whether the IFN-γ signaling via the induction CXCL9/10 was responsible 370 for the recruitment and enhanced cytotoxic response of CXCR3 + CD8 + T cells. Although IFN-γ 371 deficiency led to a significant reduction in the levels of CXCL9 and CXCL10, it did not impair the 372 recruitment of CD8 + T cells, as both WT and IFN-γ -/mice exhibited similar levels of CXCR3 + or 373 total CD8 + T cell frequency in influenza-infected lungs (Fig. 6 , E to H). These data suggest that 374 IFN-γ-independent chemokines likely compensate for the lack of IFN-γ in recruiting CD8 + T cells. 375

Our CD8 + T cell scRNAseq data show a dominant interferon signaling that correlated with 376 exuberant cytotoxic response in Cxcr3 hi CD8 + T cells. We, therefore, investigated if IFN-γ 377 regulated the cytotoxic function of CXCR3 + CD8 + T cells. We performed intracellular cytokine 378 staining to investigate the cytolytic (GzmB, Perforin) properties in CD8 + T cells. The CD8 + T cells 379 from WT and IFN-γ -/mice (influenza-infected) did not exhibit any difference in the intracellular 380 expression of GzmB and Perforin (Fig. 6 , I and J). These data suggest that IFN-γ is dispensable to 381 the recruitment and regulation of cytotoxic molecules expression in CXCR3 + CD8 + T cells during 382 influenza pathogenesis. In this study, we dissected the CD8 + T cells responses in influenza-infected lungs during the peak 387 viral (acute) and virus-cleared states. The major finding of this study is that the CD8 + T-cell 388 population recruited to the influenza-infected lungs represent significant transcriptional and 389 functional diversity, with a subset not required for viral clearance but instead driving the severe 390 lung pathology. The pathological CD8 + T-cell subset is characterized by high CXCR3 expression, 391 the enhanced cytotoxic pathway signature, and their persistence in the lungs resulting in increased 392 lung epithelium and vascular damage and the extended time of inflammatory infiltrate lung 393 consolidation. Intercepting CXCR3 with either antibody or genetic deletion prevented the 394 development of the severe influenza lung pathology, without affecting viral clearance. Thus, the 395 temporal blocking of the CXCR3 pathway could be a viable candidate for therapeutic intervention 396 that may prevent the development of significant lung injury during influenza-induced pneumonia. CXCR3 deficiency protected the CCR5 deficient mice from influenza mortality, CXCR3 420 deficiency on its own did not affect the survival of influenza-infected mice in a lethal challenge 421 model (37). In contrast, another study showed that CXCR3 deficient mice had increased survival 422 following lethal influenza challenge, and neutrophils were reported to be the primary CXCR3 423 expressing cells (38). These contrasting data necessitate further evaluating the role of CXCR3 as 424 a pathologic framework in influenza lung injury. Our model is different from those prior studies 425 because we used a severe but non-lethal model that allowed us to study the lung injury during both 426 the peak viral load and resolution. It is crucial because post-influenza complications involve 427 persistent lung injury and impaired repair with compromised lung functions after the virus is 428 already cleared (39, 40). A similar phenomenon of persistent lung injury following viral clearance 429 is observed in ongoing coronavirus disease (COVID) pandemics (41). In this context, antibody 430 blockade of CXCR3 ameliorated lung injury during the peak viral titers and led to a faster 431 resolution of post-infection lung injury. We did not detect CXCR3 + neutrophils in our model. Our 432 scRNAseq data from total lung cells demonstrated that CD8 + T cells were the only significant cell 433 type associated with CXCR3 expression during the peak viral load in the lung. 434

Interferons are crucial in regulating the anti-viral function of CD8 + T cells (42-44). In particular, 436 IFN-γ is a key regulator of the chemokines CXCL9 and CXCL10 that recruit CXCR3 + CD8 + T 437 cells to the site of infection (23). In our model, despite being a crucial regulator of CXCL9 and 438 CXCL10 chemokines, IFN-g was found to be dispensable to the recruitment of CXCR3 + or total 439 CD8 + T cells. Our data agree with prior reports showing that IFN-γ deficiency did not impact the 440 recruitment of CD8 + T cells in influenza models (45), suggesting the IFN-γ-independent 441 chemokine signaling in driving the CD8 + T cell recruitment in influenza-infected lungs. 

The lung IAV load was determined via endpoint dilution assay and expressed as 50% tissue 551 culture-infective dose (TCID50). The partial lobe of right lung from mock or IAV-infected mice 552

were homogenized in PBS with volume normalized to the lung weight and stored at -80C until use. 553

The 10-fold dilutions of lungs lysate supernatants were mixed with 3x10 3 Madin-Darby canine 554 for the downstream analysis, we discard cells with less than 200 and more than 6,000 expressed 578 genes, or the fraction of transcripts mapped to mitochondrial genes larger than 1%. The expression 579 level of each gene was normalized by using NormalizeData function of Seurat. Finally, the datasets 580 were integrated using the Seurat integration workflow. with standard settings based on the KEGG dataset. To assess differential activities of pathways 603 between different types of cells, we contrasted the activity scores for CD8 + Teff against the CD8 + 604 TN and CD8 + TCM cells by using the Wilcoxon test, and an adjusted P-value < 0.05 was used as the 605 cutoff value for significant pathways identification. 606

To determine global communications among cells, CellChat (53) Data are shown as means ± SEM, representative data of 2 experiments with n=5 per group. (G) Flow cytometry representative graphs (left) and count of CCR2 + monocytes at 7 dpi. CCR2 + monocytes were gated from CD45 + CD11b + Ly6C + cells. (H to J) Count of IFN-γ (H), Perforin (I) and Granzyme B (GzmB) (J)expressing CD8 + T cells at 7 dpi. Data are shown as means ± SEM, pooled data of 2 experiments with n=8-10 per group. (K to L) Levels of CXCL9(K) and IL-10(L) at 7 dpi, measured using BiolegendPlex kit. Data shown as means ± SEM with n=5. Oneway ANOVA with Tukey post hoc test for multiple comparison (means) was used for statistical significance in (C to L). * p< 0.05, ** p< 0.01, *** p< 0.005 and **** p< 0.001. WT and CXCR3 -/mice were mock-infected with PBS (mock) or infected with 1,000 PFUs of IAV (PR8) intranasally. At 7 dpi mice were euthanized, and lungs and BALF were aseptically collected for further analysis. (A) H&E staining of paraffin-embedded lungs sections from WT (top) mice and CXCR3 -/mice (bottom) at 7 dpi. Representative images of mock (left) and PR8-infected (right) mice with n=5 per group, images taken at X20 magnification with scale bar 100um. (B) Level of LDH in BAL of PR8 infceted WT and CXCR3 -/mice. Data shown as means ± SEM. One-was ANOVA with Tukey post hoc test was used for multiple comparisons of means between groups. * p< 0.05, *** p< 0.005 and **** p<0 .001 (C) TCID50 influenza viral load in lung homogenates of PR8-infected WT and CXCR3 -/mice. Data shown as means ± SEM. One-way ANOVA test for multiple comparisons of means between groups was used. * p< 0.05, *** p< 0.005 and **** p<0 .001

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The data is representative data of 2 independent experiments with n=5. (D) scRNAseq analysis of total lung single cells of mock and PR8-infected WT mice at 7 dpi. Violin plots showing the expression of CXCR3 ligands CXCL9 and CXCL10 in lung cells. *Mean significant difference between mock and IAV-infected mice. (E-F) Levels of CXCL9 (E) and CXCL10 (F) at 7 dpi. (G to H) Number of total (G) and CXCR3 + CD8 + T cells (H) at 7 dpi. (I-J) Expression of GzmB (I) and Perforin (J) in CD8 + T cells. (G to J) The data is representative of 2 independent experiments with n=10. One-way ANOVA with Turkey post hoc test was used for statistical significance. Data shown as means ± SEM

CD8 + Teff clusters in mock and PR8 infecetd mice at D7 and D14.The color stands for the normalized enrichment score (NES) value. (B) Top: Venn Diagram showing the shared and unique significant KEGG pathways between Cxcr3 hi and Cxcr3 low CD8 + Teff clusters in mock and PR8 infecetd mice at D7 versus Mock

The color stands for (up-regulated (red) or down-regulated (blue) in the corresponding groups. (C) Top: Venn Diagram showing the shared and unique significant KEGG pathways between Cxcr3 hi and Cxcr3 low CD8 + Teff clusters in mock and PR8 infecetd mice at D7 versus Mock, D14 versus Mock, and D14 versus D7 of E4 from GSEA; Bottom: Dot plot shows the shared significant pathways among all three comparisons. The color stands for (up-regulated (red) or down-regulated (blue) in the corresponding groups). (D) Top: Venn Diagram shows the shared and unique significant KEGG pathways between Cxcr3 hi and Cxcr3 low CD8 + Teff clusters in mock and PR8 infecetd mice at D7 versus Mock

Bottom: Dot plot shows the shared significant pathways among all three comparisons. The color stands for the normalized enrichment score (NES) value (up-regulated (red) or down-regulated (blue) in the corresponding groups)

Shared: DEGs are shared among mock, 7 and 14 dpi; D7_Mock: DEGs are shared at mock and 7 dpi; D14_Mock: DEGs are shared at mock and 14 dpi

Shared: significant pathways are shared among mock, 7 and 14 dpi; D7_Mock: significant pathways are shared at mock and 7 dpi; D14_Mock: significant pathways are shared at mock and 14 dpi

Shared or unique DEG subsets among D7 vs Mock, D14 vs Mock and D14 vs D7 in cluster E1. Shared: DEGs are shared among all three comparisons; D14vsMock_D14vsD7: DEGs are shared at mock and 7 dpi; D14_Mock: DEGs are shared at mock and 14 dpi

Shared or unique significant pathway subsets among D7 vs Mock, D14 vs Mock and D14 vs D7 in cluster E1. Shared: significant pathways are shared among all three comparisons; D14vsMock_D14vsD7: significant pathways are shared at mock and 7 dpi; D14_Mock: significant pathways are shared at mock and 14 dpi

Shared or unique DEG subsets among D7 vs Mock, D14 vs Mock and D14 vs D7 in cluster E4. Shared: DEGs are shared among all three comparisons; D14vsMock_D14vsD7: DEGs are shared at mock and 7 dpi; D14_Mock: DEGs are shared at mock and 14 dpi

Shared or unique significant pathway subsets among D7 vs Mock, D14 vs Mock and D14 vs D7 in cluster E4. Shared: significant pathways are shared among all three comparisons; D14vsMock_D14vsD7: significant pathways are shared at mock and 7 dpi; D14_Mock: significant pathways are shared at mock and 14 dpi

Shared or unique DEG subsets among D7 vs Mock, D14 vs Mock and D14 vs D7 in cluster E6. Shared: DEGs are shared among all three comparisons; D14vsMock_D14vsD7: DEGs are shared at mock and 7 dpi; D14_Mock: DEGs are shared at mock and 14 dpi

Shared or unique significant pathway subsets among D7 vs Mock, D14 vs Mock and D14 vs D7 in cluster E6. Shared: significant pathways are shared among all three comparisons; D14vsMock_D14vsD7: significant pathways are shared at mock and 7 dpi; D14_Mock: significant pathways are shared at mock and 14 dpi