key: cord-0326388-ru2u78jo
authors: Mukherjee, Ratnadeep; Singh, Diwakar Kumar; Patra, Rajkumar; Barman, Pijus Kanti; Prusty, Birendra Kumar; Thatoi, Pravat; Tripathy, Rina; Das, Bidyut Kumar; Ravindran, Balachandran
title: A novel polymorphism in nitric oxide synthase interacting protein (NOSIP) modulates nitric oxide and mortality in Human Sepsis
date: 2020-10-08
journal: bioRxiv
DOI: 10.1101/038398
sha: faaf121fc7b5bb2378ac66fbcc3bbf19a9ef15c0
doc_id: 326388
cord_uid: ru2u78jo

Nitric oxide, synthesised by three isoforms of Nitric Oxide synthases viz., nNOS by neurons, eNOS by endothelial cells and iNOS by phagocytes, performs a wide variety of biological functions in neurons, vascular endothelial cells and immune cells. Interaction between inducible nitric oxide synthase (iNOS) and Nitric oxide synthase interacting protein (NOSIP) was observed both in human monocytes and mouse macrophages and in cell free systems by biophysical methods. A novel mutation in nitric oxide synthase interacting protein (NOSIP) determined NO levels produced by human monocytes and was associated with disease severity in Sepsis patients. The study reveals NOSIP as an important regulator of inflammation by virtue of its ability to influence nitric oxide production both in mice and in humans and opens up novel avenues for therapeutic strategies against acute inflammation. While the influence of this novel NOSIP polymorphism in cardio-vascular and neuronal functions could be a subject of future investigations, its role in determining disease severity and mortality of the ongoing Covid 19 pandemic will be of immediate relevance.

The acute physiological and chronic health evaluation II (APACHE II) scoring system 68 was used to categorize the patients. Definitions of sepsis, severe sepsis, septic shock, and 69 multi-organ dysfunction syndrome (MODS) were in accordance with published 70 criteria [35, 36] . The following categories of patients were excluded from the study: endotoxemia. Blood was collected in vials containing ACD as anticoagulant (15% v/v). 84 collected blood was centrifuged at 2000 rpm for 10 minutes for isolation of plasma. 85 Isolated plasma was stored in 100 µl single-use aliquots at -80°C. 86 Assessment of nitric oxide deficiency on LPS -mediated 87 inflammation 88 8 -10 weeks old male BALB/C mice were injected with 100 mg/kg L-NAME once every 89 24 hours for inhibition of nitric oxide synthesis. To examine effect of nitric oxide 90 synthase inhibition on LPS -mediated pathology, untreated or L-NAME treated mice 91 were injected with 2 mg/kg LPS and mortality was monitored for 4 days. For comparing 92 LPS toxicity between wild -type and inos knockout mice, male animals between 8 -10 93 weeks age were injected with LPS at 15 mg/kg and either mortality was scored for 5 94 days or animals were sacrificed at 2, 6 and 12 hours to estimate cytokines in plasma. 95 Ex vivo stimulation of human and mouse peripheral blood 96 Whole blood was withdrawn from apparently healthy human donors by venepuncture or 97 from normal healthy mouse by cardiac puncture in acid citrate dextrose (ACD) 98 anticoagulant at 15% v/v. Human donors were recruited from institute students. To 99 test for cytokine expression, 50 µl whole blood was left untreated or stimulated with 100 LPS at 1 µg/ml for 2 hours in 37°C water bath along with brefeldin A (eBiosciences) at 101 1:1000 dilution. Post stimulation, cells were stained with fluorochrome conjugated 102 antibodies and analysed on a flow cytometer. 103 

Measurement of cytokines in plasma 104 Human plasma was analysed using the human 27-plex cytokine panel (Bio-Rad) 105 according to manufacturer's instructions and contained the following targets: IL-1β, 106 IL-1ra, IL -2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12(p70), IL-13, IL-15, IL-17,   107 Basic FGF, Eotaxin, G-CSF, GM-CSF, IFN-γ, IP-10, MCP-1, MIP-1α, MIP-1β, PDGF, 108 RANTES, TNF-α, and VEGF. Mouse plasma was analysed using the mouse 23-plex 109 cytokine panel (Bio-Rad) as specified by the manufacturer and contained the following 110 targets: IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12(p40), IL-12(p70), complete IMDM media containing 10% fetal bovine serum. Cells were made to adhere 134 by treatment with 10 nM Phorbol myristate acetate (PMA) for 48 hours. Following 135 adherence, cells were washed with PBS and fixed with 2% paraformaldehyde (PFA).

After rinsing with PBS, cells were permeabilized with 0.1% Triton X-100 following 137 which they were incubated in a blocking buffer containing 2% bovine serum albumin 138 (BSA) and 2% fetal bovine serum (FBS) in PBS to minimize nonspecific binding of 139 antibodies. After blocking, cells were first incubated with primary antibody followed by 140 fluorochrome conjugated secondary antibodies. Finally, cells were washed, 141 counterstained with DAPI and image was acquired in a Leica SP5 confocal microscope. 142

Imaging cytometry analysis of NOSIP expression 143 For comparing intracellular NOSIP expression between ins and del individuals, 100 µl 144 whole blood from each of 4 ins and 3 del individuals were fixed, lysed and 145 permeabilized followed by staining with anti-NOSIP antibody. After washing, the cells 146 were resuspended in PBS and acquired on an ImagestreamX imaging cytometer (Amnis 147 corp.). 20,000 events per sample were acquired for analysis. The obtained events were 148 gated and analysed by IDEAS 6.0 software. 

Total RNA was extracted from mouse or human whole blood using RNA blood mini kit 225 (Qiagen). Isolated RNA was converted to complementary DNA (cDNA) using RT 2 first 226 strand cDNA synthesis kit (Qiagen). The cDNA thus obtained was subjected to Human whole blood was stimulated for 4 hours with LPS at 1µg/ml. Following 231 stimulation, total RNA was extracted using a RNA blood mini kit (Qiagen). Total RNA 232 to cDNA conversion was done using RT 2 first strand cDNA synthesis kit (Qiagen).

Prepared cDNA was subjected to a customised qRT -PCR array (SABiosciences) using 234 RT 2 SYBR® Green qPCR Mastermix (Qiagen). Fold change over untreated control 235 was calculated using the 2 -∆∆Ct method. The obtained fold change values were used for 236 pathway enrichment analysis (Ingenuity Systems). Since binding of NOSIP to eNOS (endothelial NOS, NOS3) and nNOS (neuronal NOS, 251 NOS1) and its inhibition of their function has been reported earlier, the first objective 252 of the current study was to investigate whether NOSIP interacts with iNOS. To test for 253 direct physical interaction between the two molecules in a cell free system, we Figure S1A and B). In situ proximity ligation analysis 278 of iNOS and NOSIP in primary human monocytes further confirmed a direct physical 279 interaction between iNOS and NOSIP ( Figure S1C ). Interestingly, we observe 280 considerable nuclear localization of both iNOS and NOSIP ( Figure S1 ), which could be 281 a potential mechanism by which NOSIP inhibits iNOS. The data from (E) was fitted using a four-parameter Hill function. (G) THP-1 cells were nucleofected with empty plasmid or NOSIP shRNA plasmid by Amaxa nucleofector II using manufacturer's kit. After 48 hours, the cells were washed and incubated with or without LPS at 1 µg/ml for a further 24 hours. Total nitrate+nitrite in culture supernatant was measured by a commercial Griess assay kit. Statistical significance was assessed by two -way ANOVA following Bonferroni's post test (** p<0.01). The graph represents data from 5 separate plates performed on 2 different days. Higher NOSIP expression in humans is associated with 311 increased inflammation and susceptibility to acute inflammatory 312 diseases 313 We then wanted to test the functional significance of NOSIP mutation in host 314 inflammatory responses. Whole blood of normal human volunteers were stimulated with 315 LPS for 4 hours and gene expression was compared between ins or del subjects by a 316 custom designed qRT-PCR array ( Figure 3A ) -very broadly, expression of many of the 317 cytokine and chemokine genes in subjects with del/del genotype were higher in 318 comparison to those with Ins/Ins genotype (Cluster 4, Figure 3A ). In order to gain 319 insights into global differences in signalling pathways as a consequence of differential 320 NOSIP expression, a pathway enrichment analysis was performed. The analysis revealed 321 increased enrichment of pathways associated with inflammatory response, viz. Toll-like 322 receptor signalling, NF-κB signalling, p38 MAP kinase signalling and HMGB1 signalling 323 in individuals with del allele ( Figure 3B) . A similar enrichment analysis conducted for 324 disease associated networks revealed higher enrichment of sepsis, septic shock network 325 in Del individuals. Higher representation of networks associated with liver and kidney 326 damage, apoptosis of liver and kidney cells, and apoptosis of macrophages, phagocytes 327 and antigen presenting cells were also observed suggesting an increased risk of acute are highly prone to mortality in the cohort of sepsis patients studied (Table 2) .

Taken together, the above results clearly demonstrate that individuals homozygous 339 for the deletion allele display increased inflammatory features and are more susceptible 340 to acute inflammation induced morbidity and mortality.

NOSIP levels are associated with differential response to LPS 342 activation between circulating monocytes of mouse and human 343 origin 344 There is evidence in literature about a species-specific hierarchy in susceptibility to 345 acute inflammation [37] . Our next objective was to test whether NOSIP levels are 346 associated with species-specific variability in a host's response to inflammatory insult. A 347 comparison of intracellular iNOS and NOSIP expression between circulating monocytes 348 of human and mouse revealed significantly higher levels of iNOS ( Figure 4A ) and 349 NOSIP in human monocytes ( Figure 4B ). In concordance with the above data, we also 350 observed significantly lower levels of nitrite in plasma of sepsis pateints as compared to 351 plasma of mice injected with LPS ( Figure S3 ). We then wanted to test the functional 352 consequence of differential expression of NOSIP between mouse and human immune cells 353 upon inflammatory activation. As such, human and mouse whole blood were stimulated 354 with LPS and intracellular IL-1β and TNF-α were measured by flow cytometry. For the 355 purpose of comparison, we tested CD14(+)/Ly-6G(-) cells in mice blood with that of upon stimulation with LPS ( Figure 4C and 4E) . Interestingly, however, TNF-α levels 360 were comparable between the two species as depicted in Figure 4D and 4E.

A key point to note here is that inbred SPF mice are very different in terms of their 362 physiology from humans due to subclinical infections or vaccinations. Moreover, 363 experimentally induced endotoxemia in mice is not similar to human sepsis. However, in 364 spite of the above caveats, the above observations indicate that differential synthesis of 365 nitric oxide (as a consequence of differences in NOSIP expression) between species may 366 be in part responsible for regulating the response to acute inflammatory stimulus. The observation that increased NOSIP leads to decreased NO synthesis may be harmful 371 to host during acute inflammation led to us to investigate the role played by NO during 372 acute inflammatory disorders. In order to gain insight into the role of nitric oxide in 373 acute inflammation, a multivariate correlation analysis of plasma cytokines and nitrite 374 levels was conducted in human sepsis patients. A significant negative association was 375 observed between plasma nitrite and some of the cytokines/chemokines (Table 3) (Table 3) . From the above data, we hypothesised that absence of nitric oxide could exacerbate an 384 inflammatory insult. To investigate this, male BALB/C mice were treated with 100 385 mg/kg of Nω-Nitro-L-arginine methyl ester (L-NAME) hydrochloride once every 24 386 hours to block nitric oxide synthase activity. L-NAME is a structural analogue of 387 L-Arginine that competitively inhibits all three isoforms of nitric oxide synthase.

Untreated and L-NAME treated mice were then injected with 2 mg/kg of LPS and 389 mortality was assessed for 96 hours. Figure 5A shows comparison in LPS toxicity 390 between untreated and L-NAME treated animals clearly demonstrating that inhibition 391 of nitric oxide synthesis is detrimental to host in a mouse model of endotoxemia. This 392 was further tested by studying LPS toxicity in mice deficient in Nos2 gene. Comparison 393 of lethal dose of LPS between wild type C57BL/6 mice and Nos2 null revealed that 394 15mg/kg of LPS did not result in mortality of wild type mice, while about 84% of the 395 animals in the Nos2-/-group died at the same dose of LPS ( Figure 5B ). This confirmed 396 the hypothesis that absence of nitric oxide inducing enzyme and consequently nitric 397 oxide results in increased susceptibility to endotoxemia. To test for possible mechanisms 398 of the observed phenomenon, wild type and Nos2-/-mice were injected with LPS at a 399 dose of 15mg/kg and the animals were sacrificed at 2, 6 and 12 hours post 400 administration and plasma cytokines were quantified. The heatmap shown in Figure   401 5C reveals significantly higher levels of IL-1β in mice lacking the Nos2 gene. However, 402 plasma levels of TNF-α were comparable in both ( Figure 5C ), suggesting that IL-1β 403 could be playing a more central role in poor disease outcome in endotoxemia/sepsis and 404 that NO is a key molecule regulating NLRP3 mediated inflammasome pathway. Very 405 similar observations on the role of nitric oxide on IL-1β in endotoxemia has been 406 reported earlier [34] . In order to obtain a more global understanding of the effect of 407 Nos2 deficiency in LPS induced inflammation, we performed a principal component 408 analysis (PCA) of the measured cytokines. The scatterplot depicted in Figure 5D 409 clearly shows the best separation of wild type and Nos2-/-along the third principal 410 component. This is also evident from the scores on principal component 3 (PC3), which 411 shows a clear dichotomy between wild type and Nos2-/-mice ( Figure 5E ). Analysis of 412 the contribution of individual cytokines along PC3 revealed IL-1β to be the highest 413 positive contributor towards the observed differences between wild type and Nos2-/-414 mice ( Figure 5F ). Moreover, we also observed significantly higher levels of IL-5 in 415

Nos2-/-mice along with a concordant decrease in IL-12 and IFN-γ levels ( Figure 5C 416 and F). This indicates a possibility of a T-helper type2 skewed response in the Nos2-/-417 mice.

To summarize, the above findings demonstrate that absence of Nos2 is deleterious to 419 host in a mouse model of acute inflammation that involves increased production of 420 IL-1β. Figure 6 summarizes the principal findings of this study. [39, 40] . Curiously however the role of NOSIP in regulating 429 iNOS and its consequence in mammalian immune system has not been investigated. We 430 provide unequivocal biochemical and biophysical evidence (apart from in situ 431 microcopic and functional proof) for interaction between iNOS and NOSIP using a cell 432 free system using recombinant protiens, unlike previous investigations on eNOS/nNOS 433 and NOSIP, that mostly utilized yeast two-hybrid screens and chromatin 434 immunoprecipitation assays [26, 27] . 435 Nitric oxide (NO) is synthesised by many cell types during host response to 436 pathogens and injury [4] . The principal enzyme responsible for nitric oxide production 437 in immune cells during inflammation is inducible nitric oxide synthase (iNOS or NOS2). 438 Role of nitric oxide in endotoxemia/sepsis has been very contradictory in reported cecal-ligation-and-puncture induced sepsis, was found to be dependent on iNOS activity 449 expressed by FRCs [41] . In a similar sepsis model, iNOS-dependent upregulation of 450 cGMP and subsequent activation of TACE was found to protect against organ 451 injury [42] . Other studies in animal models have also documented possible protective 452 role of NO in endotoxemia [43] .

The current manuscript presents several novel findings on modulation of iNOS 454 activity and nitric oxide biology in the context of host response by Nitric oxide synthase 455 interacting protein (NOSIP). Several years ago NOSIP was discovered to interact with 456 eNOS and inhibit its function by sequestering it in Golgi complex [27] . Subsequently, 457 interaction of NOSIP with nNOS was also reported [26] . Curiously however, till date 458 the role of NOSIP and its with iNOS and regulation of nitric oxide production by 459 immune cells has not been investigated -the present study fills this lacuna. Physical Ligation of LPS-CD14 complex (not shown for sake of simplicity) to membrane TLR4, leads to activation of iNOS along with IL-1β, that is secreted from the cell. (Left) In the absence of NOSIP protein, iNOS is available to catalyze the conversion of L-Arginine to nitric oxide (NO), which then inhibits IL-1β production, thereby leading to less IL-1β secretion. (Right) When NOSIP is present, it binds to iNOS and inhibits its ability to convert L-Arginine to NO. Less nitric oxide leads to increased production and secretion of IL-1β.

interaction between NOSIP with iNOS were demonstrable in immune cells as well as in 461 cell free systems using recombinant proteins. More significantly, NOSIP emerged as a 462 key molecule regulating iNOS function and release of nitric oxide. More critically a four 463 nucleotide deletion upstream of first exon of NOSIP gene was found to be associated 464 with increased intracellular expression of NOSIP and higher risk of mortality due to interactions which could modulate nitric oxide levels and consequently inflammation.

More significantly such small molecules could also be useful in regulating nitric oxide 471 production by nNOS and eNOS in neuronal and endothelial cells respectively.

An inverse association of plasma nitrite with inflammatory cytokines was observed 473 in human sepsis and mouse model of endotoxemia, suggesting a reciprocal regulation of 474 nitric oxide and inflammatory mediators. Species-specific differences in nitric oxide 475 synthesis have been well documented in literature. A major issue of debate in literature 476 has been differences in NO production among species, most notably between human and 477 mouse [44, 45] . It has been reported earlier that human monocytes and macro-phages 478 produce significantly lower amounts of NO than their mouse counterparts [46] . Whether 479 such differences translate into differences in susceptibility to acute inflammation remains 480 unclear and is one of the key questions addressed in the present study. The results show 481 a clear positive association between nitrite oxide production and resistance to LPS 482 mediated inflammation, suggesting that a given species' ability to tolerate inflammatory 483 insult is, at least in part, dictated by its ability to produce nitric oxide. This conclusion 484 may have important bearing on a study published earlier that demonstrated that 485 proteins in serum rather than intrinsic cellular differences may play a role in regulating 486 variations in resilience to microbe-associated molecular patterns between species [37] . It 487 is apparent that identification of such proteins may open up new areas of targeted 488 therapy. However, given the large number of proteins in serum, this is an arduous task. 489 The observations made in the current study could help in narrowing down the search by 490 initially probing enzymes involved in nitric oxide biosynthesis.

Another important finding of the present investigation was demonstration of 492 increased IL-1β production in mice lacking a functional iNOS gene (during 493 inflammatory activation), suggesting that protective effect of nitric oxide in 494 endotoxemia is mediated through inhibition of IL-1β synthesis -more so since TNF-α 495 levels remained comparable in wild type and iNOS deficient mice. This is particularly 496 interesting in the light of an earlier study that demonstrated no difference in LPS 497 toxicity between wild type and TNF-α knockout mouse [47] . On the other hand, mice 498 deficient in IL-1β converting enzyme were relatively resistant in comparison to wild 499 type mice when challenged intraperitoneally with endotoxin [48] . These observations 500 suggest a central role played by IL-1β (inflammasome pathway) in pathogenesis in 501 endotoxemia/sepsis. Similar to nitric oxide, species specific dichotomy in terms of IL-1β 502 production was observed between human and mouse circulating immune cells that 503 negatively correlated with nitric oxide production, further demonstrating IL-1β as a 504 central pathogenic hub in endotoxemia.

Despite advances in both understanding of basic biology as well as better critical 506 care support, mortality due to sepsis remains unacceptably high. As of now, no effective 507 therapy exists to combat sepsis. Given the protective role played by nitric oxide in 508 sepsis, it is tempting to suggest administration of nitric oxide donors for clinical 509 management of sepsis. Indeed, several studies have attempted NO supplementation in 510 sepsis, and a systematic review and meta-analysis of such studies showed that this line 511 of therapy could be promising [49] . However, because of the extremely short half-life of 512 19/28 nitric oxide, NO donors require administration on a regular basis, thus potentially 513 raising the cost of treatment. In this context, identification of NOSIP as a key regulator 514 of NO synthesis immediately opens up exciting possibilities for developing targeted 515 therapy. Designing inhibitors of NOSIP could lead to increased NO production which, 516 in turn, could control hyper-inflammation observed in sepsis. Moreover, since nitric 517 oxide acts as a potent vasodilator and an inhibitor of platelet aggregation, the 518 implications of developing inhibitors against a protein that modulates its synthesis are 519 quite attractive as a therapeutic strategy in cardiovascular diseases. However, owing to 520 the novelty of NOSIP, extreme caution should be exercised before designing such 521 therapies as the overall function of NOSIP in other cellular processes need to be 522 evaluated first, particularly their effect on eNOS and nNOS mediated biological 523 activities -a recent finding reported a crucial requirement of NOSIP during 524 neurogenesis in both mice and Xenopus [50] . Since increased NOSIP expression appears 525 to render a host more susceptible to acute inflammation from an evolutionary 526 perspective, it would be interesting to investigate differences in organ specific NOSIP 527 expression between species and strains of animals.

Finally, from a translational perspective the role of this novel NOSIP polymorphism 529 viz., (subjects with homozygous 'del' allele being more prone for inflammation), in 530 determining disease severity as well as mortality of the ongoing Covid 19 pandemic will 531 be of immediate relevance to be used as a biomarker in Covid 19 infections -host 532 inflammation is a key factor in pathogenesis of Covid 19 infections [51] and use of 

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The authors thank Dr. Dileep Vasudevan, Institute of Life Sciences for his assistance in 536 cloning and purification of iNOS and NOSIP proteins and Dr. Narottam Acharya,