Sex-dependent effects of peptidylarginine deiminases on neutrophil function and long-term outcomes after spinal cord injury

Sex-dependent effects of peptidylarginine deiminases on neutrophil function and long-term outcomes after spinal cord injury | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Sex-dependent effects of peptidylarginine deiminases on neutrophil function and long-term outcomes after spinal cord injury Shelby K. Reid , Ashley V. Tran , Miranda E. Leal-Garcia , Sachit Devaraj , Mustafa Ozturgut , View ORCID Profile Dylan A. McCreedy doi: https://doi.org/10.1101/2025.05.08.652924 Shelby K. Reid a Texas A&M Institute for Neuroscience, Texas A&M University , 3474 TAMU, College Station, Texas, 77843, USA b Department of Biology, Texas A&M University , 3258 TAMU, College Station, Texas, 77843, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ashley V. Tran b Department of Biology, Texas A&M University , 3258 TAMU, College Station, Texas, 77843, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Miranda E. Leal-Garcia b Department of Biology, Texas A&M University , 3258 TAMU, College Station, Texas, 77843, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Sachit Devaraj b Department of Biology, Texas A&M University , 3258 TAMU, College Station, Texas, 77843, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Mustafa Ozturgut b Department of Biology, Texas A&M University , 3258 TAMU, College Station, Texas, 77843, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Dylan A. McCreedy a Texas A&M Institute for Neuroscience, Texas A&M University , 3474 TAMU, College Station, Texas, 77843, USA b Department of Biology, Texas A&M University , 3258 TAMU, College Station, Texas, 77843, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Dylan A. McCreedy For correspondence: dmccreedy{at}bio.tamu.edu Abstract Full Text Info/History Metrics Preview PDF Abstract Traumatic spinal cord injury (SCI) initiates an influx of peripheral immune cells to the spinal cord parenchyma that compound tissue damage and restrict functional recovery. Neutrophils infiltrate the spinal cord within the first day after injury, releasing extracellular traps (NETs) comprised of decondensed DNA, modified histones, and granule enzymes, that can worsen tissue damage. Peptidylarginine demininases (PADs), particularly PAD4, have been indicated as mediators of NET formation by facilitating the decondensation of nuclear chromatin via histone citrullination. Though PADs have been shown to be regulated by sex hormones, sex-differences in PAD regulation of neutrophil function in the context of CNS injury have yet to be explored. In this work, we investigated the role of PADs in recovery after SCI using Cl-amidine, a pan-PAD inhibitor. Strikingly, Cl-amidine treated mice exhibited sex-dependent changes to motor function, body weight, and white matter sparing after SCI. Acutely, Cl-amidine treated mice had reduced NET accumulation in the blood and decreased spinal cord neutrophil granularity. Analysis of publicly available scRNA-seq data revealed that female bone marrow neutrophils exhibited elevated Padi4 expression relative to their male counterparts. We then utilized Padi4 knockout ( Padi4 -/- ) mice to assess the role of PAD4 in long-term recovery of male and female mice after SCI. While we observed no changes in motor recovery, a sex-dependent effect on tissue sparing was observed with Padi4 deficiency. These data are the first description of sex differences in PAD-mediated neutrophil function after SCI and highlight the importance of inclusion of both sexes in pre-clinical research. Introduction Traumatic spinal cord injury (SCI) is a highly debilitating condition marked by loss of motor function and sensation below the level of injury. The primary SCI event initiates a cascade of inflammatory processes that exacerbate tissue loss and impair long-term functional outcomes 1 , 2 . Breakdown of the blood-spinal cord barrier (BSCB) accompanies an influx of peripheral immune cells into the injured spinal cord within minutes to hours after SCI 3 , 4 . In the early hours after injury, neutrophils swarm to the injury site en masse , peaking in number in the first day after injury in rodent models of SCI 5 – 9 . While neutrophils have been primarily characterized as harmful in SCI, their precise role in SCI pathology and their activities in the injured spinal cord remain largely unknown. Neutrophils are the most common circulating white blood cell population in humans and serve as patrolling first-responders to infection and injury. As critical mediators of host defense, neutrophils are equipped with a suite of effector functions including phagocytosis, release of reactive oxygen species, cytokine production, degranulation, and extracellular traps (ETs). Neutrophil extracellular traps (NETs) are a specialized effector function wherein the neutrophil will decondense its chromatin, decorate the DNA with cytotoxic granule proteins, and extrude it from the cell body into the extracellular space. ETs’ web-like structure serves to contain and kill pathogens; however, ETs can also be released in response to non-pathogens such as crystals, immune complexes, and tissue injury, causing collateral damage to host tissue 10 – 13 . CNS injury has been shown to stimulate ETosis, often leading to worse long-term outcomes 14 – 23 . In rodent models of SCI, ETosis has been linked to exacerbation of acute inflammation and worse recovery of long-term motor function 24 – 28 . However, sex as a biological variable has yet to be considered in ETosis after SCI; thus, any sex differences in the mechanisms of ET formation or response to treatment remain unknown. While PAD4 citrullination of histones is thought to be the primary mediator of chromatin decondensation in NETosis, other PADs are also expressed in neutrophils and may play a redundant role to PAD4 11 , 29 – 32 . In the present study, we assessed the role of PAD activity and PAD4 in ET formation after SCI using pharmacological and genetic approaches with a particular focus on sex as a biological variable. We found that PAD activity has sex-dependent effects on long-term outcomes following SCI including motor recovery, weight loss, and white matter sparing. Acutely, we show that PADs mediate neutrophil responses in the injured spinal cord. Furthermore, using a PAD4 knockout, we show that PAD4 regulates long-term tissue damage after SCI in a sex-dependent manner. Together, these data show sex-dependent roles for PADs in acute inflammation and long-term outcomes following SCI, highlighting the necessity of including sex as a biological variable in neurotrauma research. Results PAD inhibition alters long term SCI outcomes in a sex-dependent manner To assess whether PADs play a role in long-term outcomes following SCI, while also considering sex as a biological variable, we utilized a pharmacological approach to broadly inhibit PAD activity. Cl-amidine is a potent, irreversible small molecule pan-PAD inhibitor frequently used in animal studies to abrogate ET formation 33 , 34 ( Fig 1A ). Cl-amidine administration had no appreciable effect in the BMS main score ( Fig 1B ); however, sex-dependent effects on motor recovery were evident in the BMS subscore ( Fig 1C , Time x Sex x Treatment interaction p=0.01165) with male mice trending towards improved subscores and female mice trending towards reduced subscores. Body weights of the mice were assessed throughout the course of the study to monitor the health of the animals as a sharp reduction in body weight can be indicative of poor health and require euthanasia of the animal. We observed a sex-dependent effect of Cl-amidine on body weight retention ( Fig 4D , Time x Sex x Treatment, p=0.032) with male mice trending towards greater body weight retention and female mice trending towards greater weight loss. Mice exhibiting a BMS score ≥ 4 by 28 dpi were assessed via the horizontal ladder rung walking test (LRWT). However, no statistical differences were found between groups in percentage stepping or cumulative errors ( Fig 1E-F ). Download figure Open in new tab Figure 1: PAD inhibition with Cl-amidine alters long-term motor recovery and weight loss after SCI in a sex-dependent manner. (A) Cl-amidine (50 mg/kg) was administered intraperitoneally immediately following moderate T9 contusion. Functional recovery was assessed for 5 weeks following injury via the Basso Mouse Scale (BMS) and the horizonal ladder rung walking test (LRWT). (B-C) While no significant differences between groups were observed via the (B) main BMS score, (C) BMS subscores were differentially altered between sex and Cl-amidine treated groups across time (C: Time x Sex x Treatment, p= 0.0116; 3-way RM ANOVA, n=6-10/treatment/sex). (D) Body weight retention over time following SCI was differentially altered between sex and Cl-amidine treated groups. (Time x Sex x Treatment, p=0.0320; 3-way RM-ANOVA, n=6-10). (E-F) At 34 dpi, mice with BMS ≥ 4 were assessed via LRWT. No statistically significant differences in (D) percentage of plantar stepping or (E) cumulative error across trials were observed (2-way ANOVA, n=3-5). Data shown as mean ± SEM. *p<0.05, **p<0.01. After tissue collection at 35 dpi, we examined white matter tissue sparing to assess the long-term tissue damage across the lesion site. We found a sex-dependent effect of Cl-amidine treatment on white matter tissue sparing ( Fig 2A , Treatment x Sex, p=0.040) and lesion length ( Fig 2B , Treatment x Sex, p=0.015). Specifically, we observed greater lesion length, as well as a strong trend (p=0.059) towards reduced white matter sparing, in Cl-amidine treated female mice relative to vehicle controls. No differences were observed in male mice. No effect of treatment or sex was observed for white matter sparing at the lesion center ( Fig 2C ). Together, these data demonstrate an interesting sex-dependent effect wherein female mice appear to be adversely impacted by PAD inhibition after SCI while male animals are either positively or not at all impacted by the same treatment. Download figure Open in new tab Figure 2: PAD activity alters long-term tissue damage after SCI in a sex-dependent manner. White matter tissue sparing in the spinal cord was assessed at 35 dpi. (A) Across the lesion, tissue sparing was differentially affected by sex and Cl-amidine treatment (3-way RM ANOVA, Treatment x Sex p=0.0403. Data were disaggregated by sex and performed 2-way ANOVA with Tukey’s post-hoc ; n=6-9/sex/treatment). (B) Lesion length was similarly altered by treatment and sex (2-way ANOVA, Sidak’s post-hoc , n=6-9) with Cl-amidine increasing lesion length in female mice. (C) No sex or treatment effects were observed for white mater sparing at the lesion center (2-way ANOVA). Tissue images are representative of group means. Data shown as mean ± SEM. *p<0.05, **p<0.01. PAD inhibition reduces ET accumulation and neutrophil proportion acutely after SCI To elucidate potential mechanisms behind the sex-dependent effects we observed in long-term recovery, we assessed Cl-amidine treated mice at 1 dpi, the peak of neutrophil infiltration and NET formation in the spinal cord 28 . We first quantified overall levels of ET complexes in cell-free supernatant of the spinal cord homogenate via capture ELISA. At 24 hpi, Cl-amidine treated mice had reduced ET-complex load in the blood ( Fig 3A ) with a similar trend in the spinal cord ( Fig 3A ). Download figure Open in new tab Figure 3: PAD inhibition with Cl-amidine reduces ET trap formation and alters immune cell response post-SCI. Immediately following moderate T9 contusion (0 hpi), Cl-amidine (50 mg/kg) was administered via intraperitoneal injection. Tissues were collected at 24 hpi. (A) Cl-amidine administration reduced ET complex accumulation in the blood compared to vehicle controls. A similar strong trend was observed in the spinal cord (Unpaired Student’s t-test, p SpinalCord =0.0779). (B-D) Percentage of neutrophils that were CitH3+ were assessed via flow cytometry. (B) Blood neutrophils showed no differences across groups while (C) spleen neutrophils from Cl-amidine treated animals exhibited a strong trend towards reduced CitH3 accumulation. (D) No differences were observed in CitH3+ neutrophils in the spinal cord (2-way ANOVA, Sidak’s post-hoc) . (E) Cl-amidine treatment differentially altered spinal neutrophil granularity by sex, (Treatment x Sex, p=0.002065. Grubbs’ test, 2-way ANOVA, Sidak’s post-hoc ). ( F) A strong trend towards reduction in the neutrophil proportion of CD45+ immune cells in the spinal cord was observed in female Cl-amidine treated mice (Treatment: p=0.008458; 2-way ANOVA, Sidak’s post-hoc ). (G-H) No treatment group differences were observed in total number of neutrophils, though females were observed to accumulate fewer neutrophils in the spinal cord at 24 hpi (G: 3-way RM ANOVA, Tukey’s post-hoc . H: 2-way ANOVA). Data shown as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001. Download figure Open in new tab Figure 4: Intraspinal neutrophil PAD expression differs by sex. (A) UMAP of scRNAseq data for male and female bone marrow neutrophils. Male neutrophils (black) are identified by the expression of Ddx3y and female neutrophils (red) are identified by the expression of Xist. Neutrophils with no detectable expression of Ddx3y or Xist are shown in grey. (B-C) Comparison of Padi4 (B) and Padi2 (C) transcript levels between male (Ddx3y + /Xist - ) and female (Xist + /Ddx3y - ) neutrophils. Mann-Whitney U test. ****p<0.0001. Although neutrophils make up the bulk of infiltrating peripheral immune cells at 24 hpi, we used flow cytometry to verify cell-type specificity of NET-inhibition. While blood neutrophils exhibited no differences across groups ( Fig 3B ), splenic neutrophils showed a strong trend towards a reduced percentage of CitH3 + neutrophils in Cl-amidine treated animals, irrespective of sex ( Fig 3C ). Cl-amidine did not significantly alter the percentage of CitH3 + intraspinal neutrophils ( Fig 3D ), suggesting that PAD inhibition may affect other neutrophil activities. The flow cytometry side scatter (SSC) measurement reflects the structural complexity of the immune cell and can indicate the granularity of the cells’ contents as well as cell membrane roughness accompanying cytoskeletal reorganization 35 – 37 . In Cl-amidine treated mice, we observed reduced neutrophil granularity (p=0.0376), which was particularly pronounced in female mice ( Fig 3E , p=0.045). Cl-amidine treated females also showed a trend towards reduced neutrophil proportion out of CD45 + immune cells ( Fig 3F , p=0.064). Interestingly, when absolute neutrophil numbers were assessed across the SCI lesion via IHC, no treatment effects were observed in either sex, though female animals accumulated notably fewer neutrophils than their male counterparts ( Fig 3G-H ). Together, these data show that PAD inhibition alters neutrophil activity in the injured spinal cord in a sex-dependent manner and that neutrophil response to SCI differs by sex. PAD gene expression in neutrophils differs by sex To determine which PADs are expressed by neutrophils, we performed an independent analysis of publicly available single cell RNA sequencing (scRNAseq) data from male and female bone marrow neutrophils 38 . Male and female neutrophils were identified by the presence of transcripts for the sex-specific genes Ddx3y and Xist, respectively. Interestingly, we found that bone marrow neutrophils from females had higher transcript levels for Padi4 relative to males. A similar trend was observed for Padi2 . Transcript levels for other PAD genes were minimally detected. Our findings indicate that PAD4 is most prominent in neutrophils and that expression of Padi 4 differs between female and male neutrophils. PAD4 regulates tissue sparing after SCI in a sex dependent manner To assess the specific role of PAD4 in long-term functional outcomes after SCI, we utilized a global knockout of Padi4 ( Padi4 -/- ) and assessed motor recovery for 35 dpi ( Fig 5A ). Surprisingly, no differences were observed between Padi4 -/- mice and their wild-type (WT) littermate controls in body weight retention or BMS score by 35 dpi ( Fig 5B-C ). However, examination of white matter sparing at the SCI lesion epicenter ( Fig 5E-F ) showed a significant interaction between sex and genotype, indicating that PAD4 may differentially affect tissue sparing after SCI in a sex-dependent manner. There was also a strong trend (p=0.053) towards reduced white matter sparing at the lesion epicenter in female Padi4 -/- mice relative to WT controls. These data indicate that while Padi4 - deficiency has no apparent effect on motor recovery after SCI, PAD4 may differentially affect sparing of myelinated spinal cord tissue in male and female animals. Download figure Open in new tab Figure 5: PAD4 alters tissue sparing after SCI in a sex-dependent manner. (A) Functional motor recovery was assessed for 5 weeks after moderate contusion SCI via the Basso Mouse Scale (BMS). (B) No differences in weight loss were observed between genotypes. (3-way RM ANOVA, n=6-8/sex/genotype). (C) No differences were observed in BMS score (3-way RM ANOVA, n=6-8/sex/genotype). (D-E) Spinal cord tissue centered on the lesion site was assessed for white matter tissue sparing. While no overall differences were observed (D) across the lesion, we observed a significant interaction between sex and genotype regarding white matter sparing at the (E) lesion epicenter (D: 3-way RM ANOVA (n=6-8/sex/genotype. E: 2-way ANOVA, Sidak’s post-hoc ). Tissue images are representative of group means. Data shown as mean ± SEM. ETs are released by neutrophils (among other cell types) in the injured spinal cord 24 , 39 , 40 , and PAD4 has been considered the canonical contributor to histone citrullination in the process of ETosis. To determine if PAD4 is involved in ETosis acutely after SCI, we assessed the abundance of ETs in the spinal cord lesion site and peripheral blood of Padi4 -/- mice and their wild-type littermates in a contusion model of SCI ( Fig S1A ). In Padi4 -/- mice, ET complex deposition was reduced in the spinal cord at 24 hpi ( Fig S1B ). Since neutrophils are most numerous in the spinal cord acutely after injury, we assessed NETs in vivo by flow cytometry at 24 hpi. Blood neutrophils from Padi4 -/- mice had significantly less intracellular histone citrullination than WT controls ( Fig S1C ) and intraspinal neutrophils exhibited a similar trend ( Fig S1D ). Interestingly, Padi4 -/- neutrophils from theblood had significantly higher levels of extracellular myeloperoxidase (MPO), potentially indicating degranulation, than WT controls ( Fig S1E ), which was not observed in the spinal cord ( Fig S1F ). Together, these data indicate that PAD4 mediates NET formation in SCI. Discussion Extracellular traps are a double-edged sword: not only can they harm potential invaders, but also host tissues. Thus, it may not be advantageous for all cells to undergo ETosis; indeed, only up to about 25% of neutrophils will choose NETosis when faced with infection 41 . This balancing act—a cell’s propensity to choose ETosis over other potential effector functions—is dependent on many factors including pathogen size 42 , cell maturity 43 , 44 , age 45 – 48 , and sex 47 , 49 – 54 . Sex-differences in ETosis have not been widely studied; however, ETosis is thought to be directly influenced by sex hormones 49 , 50 , 54 – 57 . Furthermore, estrogen can directly stimulate expression of Padi4 and Padi2 , while androgens suppress their expression 58 – 63 . Despite these factors, many studies of ETosis do not include both sexes and those that do include both sexes often do not disaggregate the data by sex for subsequent statistical analyses. To the best of our knowledge, we have for the first time elucidated sex differences in the role of PAD activity on the acute inflammatory response following SCI. Interestingly, Cl-amidine administration altered the properties of neutrophils in the injured spinal cord in a sex-dependent manner. Female Cl-amidine-treated mice were observed to have reduced neutrophil proportion and neutrophil granularity compared with vehicle-treated controls. While we did not observe any treatment-dependent differences in total neutrophil accumulation across the lesion, we did observe that male animals had significantly higher neutrophil counts compared to females independent of treatment. These data indicate that composition of myeloid cell populations present after SCI may be both PAD- and sex-dependent, potentially proceeding via different mechanisms. Therefore, therapeutic interventions designed to target ETosis and ET-mediated damage must take sex-differences into account. A common observation in both the laboratory and the clinic is that after neurotrauma, females recover better— or at least, differently—than their male counterparts 64 – 67 . Acute inflammation after SCI likewise differs in a sex-dependent fashion, which leads to alteration in long-term recovery outcomes 68 – 71 . As we observed changes to the acute inflammatory profiles of both PAD-inhibited animals, we assessed long term function by sex in both groups. Interestingly, broad PAD-inhibition with Cl-amidine resulted in sex differences body weight retention, motor recovery, and tissue sparing across the lesion. While Cl-amidine treated male mice trended towards improved long-term outcomes, Cl-amidine treated female mice were adversely affected. A potential explanation for this difference is that Cl-amidine administration inhibits PAD2, which is known to contribute to myelination and oligodendrocyte development 72 . The mechanism underlying the selective vulnerability of female animals to PAD inhibition after SCI remains a question for future study. PAD4 has been heavily implicated in ET formation and is considered the primary enzyme responsible for histone citrullination in the canonical pathway of ET formation 73 – 75 . However, PAD4-independent NET formation has been reported 73 , 76 – 82 . PAD2 is also expressed in neutrophils can mainly be found in the cytosol, though it can relocate to the nucleus in response to calcium signaling 29 , 83 , 84 . While PAD2 is not required for ETosis 85 , 86 , recent studies have indicated that PAD2 may play a redundant role to PAD4 in ET formation 30 , 31 , 58 , 87 – 90 . Here, we confirm that Padi4 levels differ between male and female neutrophils, at least naïve bone marrow populations. Padi4 deletion in our model of injury did not significantly alter body weight, or motor recovery following SCI. However, white matter tissue sparing displayed a sex and genotype-dependent effect at the lesion epicenter. We also found that PAD4 is responsible for a portion of ETosis in SCI and that PAD4 mediates NETs formation in both the blood and spinal cord following injury. Interestingly, we also saw an increase in extracellular MPO on neutrophils in the blood, perhaps indicating that—without PAD4, neutrophils may choose to undergo degranulation instead of ETosis. Surprisingly, pan-PAD inhibition with Cl-amidine had no effect on NET formation (CitH3 + neutrophils), albeit overall ET levels were reduced. It is possible that broad inhibition of PADs primes neutrophils for PAD-independent NETosis. In the present study, we demonstrate sex differences in PAD-mediated acute inflammation and long-term recovery in a mouse model of SCI. Immune cell accumulation in the spinal cord was PAD and sex-dependent, with PAD activity affecting neutrophil proportion in female mice only. Long-term functional and tissue sparing outcomes after injury were altered in a sex- and PAD-dependent manner. These results suggest that while PADs mediate ETosis, some PAD activity is required for optimal recovery from SCI in female animals, underlining the cruciality of assessing sex differences at the pre-clinical stage. Methods Study Design The objective of this study was to determine whether PAD4 contributes to acute extracellular trap formation and the progression of pathology (tissue sparing, motor recovery) after spinal cord injury with a focus on unveiling any sex-dependent differences. All procedures were carried out in compliance with Texas A&M University Institutional Animal Care and Use Committee guidelines (approval numbers 2019-0045 and 2021-0340). Mice C57Bl/6J mice (3-6 months) obtained from Jackson Laboratory and maintained in house were used for all pharmacological studies (#000664). For genetic studies, B6.Cg- Padi4 tm1 . 1Kmow /J ( Pad4 - , #:030315) were obtained from Jackson Laboratory and maintained in house. Pad4 -/- mice lack exons 9 and 10 essential to the PAD4 active site as well as 4 exons related to Ca 2+ binding 91 . Animals were housed in conventional 12-hr light-dark cycle with food and water accessible ad-libitum in cages containing 2-5 mice. Following surgery, male mice were singly housed to prevent incidents of aggression and were given huts for enrichment while female mice were housed together in groups of 2-3 per cage. Spinal Cord Injury Surgery Mice were anesthetized with 2% isoflurane for 10 minutes prior to surgery and kept under isoflurane for the entirety of the procedure. Following toe pinch to ensure deep anesthetization, mice were shaved and prepared with betadine and 70% ethanol to sterilize the incision area. The spinal column over the thoracic vertebral level nine (T9) lamina was exposed, the lamina was removed, and the spinal column was clamped at T8 and T10. A 60 kDyne injury with a 1 second dwell time was applied to the exposed spinal cord at T9 via an Infinite Horizons / Precision Systems and Instrumentation impactor. Superficial muscle was closed with a 5.0 suture and 50 µL of bupivacaine was administered topically to mitigate post-operative pain. The skin was closed with wound clips and given 1.0 mL of subcutaneous saline to prevent dehydration. Mice were allowed to recover in a home cage warmed on a heating pad until they recovered from anesthesia. Post-Operative Care Mouse bladders were manually voided twice per day for the entirety of the experiment. To prevent urinary tract infection and dehydration, mice were administered Cefazolin (120 mg/kg in ∼1 mL of saline) and 1.0 mL of saline daily for 10 days. Weights were assessed daily for 10 days and weekly thereafter for the entirety of the study. Cl-amidine Administration To inhibit PAD activity pharmacologically, Cl-amidine (Sigma-Aldrich) was utilized. Immediately following the conclusion of SCI surgery, Cl-amidine was administered via intraperitoneal injection at 50 mg/kg while control animals received vehicle only. For Figures 3.3A and 3.3C, Cl-amidine was dissolved in 100% DMSO. For all other figures, 0.5% DMSO in saline was used as the delivery vehicle. Experimenters were blinded to treatment groups and treatment groups were randomized prior to the study. Motor Recovery Basso Mouse Scale Following surgery, mice were assessed at 1 and 3 dpi and weekly thereafter for 35 dpi via the Basso Mouse Scale (BMS). Assessment was completed at least 1 hour post bladder voiding and carried out in room mice were housed in. BMS assessment was completed by 2 independent raters during a 4 minute period in which the mouse was allowed to roam freely in a clear acrylic open field with a mirror placed underneath. Scores were averaged between the independent raters. Animals with BMS > 2 at 1 dpi were excluded from the study. Ladder Rung Walking Test Mouse Horizontal Ladder (Maze Engineers) was used to assess coordinated motor function in mice that had achieved BMS ≥ 4 by 28 dpi. Ladder was mounted across an acrylic bin equipped with strip LED lighting to illuminate the walkway. A ramp at the end of the ladder extended into the mouse’s home cage. Mirrors were mounted at an approximately 45° angle to the walkway such that the mouse was simultaneously visible from beneath and the sides. A GoPro Hero9 was mounted below the walkway to capture footage in 4K at 60 FPS using the Linear FOV. Mice were acclimated to the ladder and ramp in their home cage for 3 min, then placed on the side of the ladder opposite to their home cage and allowed to cross and descend a ramp into their home cage. Mice were allowed to rest for 2 minutes in home cage between trials. This process was repeated until the mouse voluntarily exited the ladder. Mice were trained for 3 days prior to final assessment, which took place at 35 dpi. Mice were assessed for 5 passes. Videos were assessed by a blinded rater 92 . Briefly, each step was scored as plantar, toe, skip, slip, miss, or drag for both left and right hind paws. Percent stepping was quantified as Plantar + Toe steps / All steps x 100. Stepping errors were quantified as total Slips + Skips + Miss + Drag / number of passes. Tissue Processing Histology Tissues were collected as previously described 28 . Briefly, mice were administered a lethal dose of 2.5% Avertin then transcardially perfused with 25 mL of cold 1X PBS followed by 25 mL of cold 4% paraformaldehyde to fix tissues. Spinal columns were then dissected and left in 4% paraformaldehyde to fix overnight. Spinal cords were then dissected and cryoprotected in 30% sucrose for 2 days. Following this, 4 mm of spinal cord was mounted in OCT and cut in 25 µm sections, then stored at -80C. Flow Cytometry / ELISA Tissues were collected as previously described 28 . Briefly, mice were administered a lethal dose of 2.5% Avertin via intraperitoneal injection. Following euthanasia, blood was drawn from the heart in an EDTA coated 1 mL syringe using a 25 G needle. The needle was removed from the syringe and blood was ejected into a chilled microcentrifuge tube (MCT) then diluted with an equal volume of 2.5 mM EDTA in HBSS. MCT was inverted 8-10 times to mix and immediately centrifuged at 1350 G for 5 min at 4C. Supernatant was transferred to a fresh chilled MCT and centrifuged (1350 G, 5 min, 4C), then aliquoted and stored at -80C for ELISA. Pellet was transferred to 10 mL of ice-cold HBSS containing 50 µL of 0.5 M EDTA and kept on ice. Spinal cord (5 mm) was rapidly dissected and placed into 300 µL of ice-cold RPMI and kept on ice. Spleen was collected and connected fat was removed. Excess moisture was removed with a Kim Wipe and the spleen was weighed before being transferred to 300 µL of ice-cold RPMI. Flow Cytometry Cells were treated as previously described (Reid et al ., 2025). All steps were taken on ice and with ice-cold solutions. Briefly, cells underwent RBC lysis (BD PharmLyse solution, 1:10 dilution in water, BD Biosciences). Blood was incubated for 6 min with 10 mL of solution while spinal cord and spleen were incubated for 5 min with 1 mL of solution. Samples were subsequently diluted with NDS-FACS buffer (2% Normal Donkey Serum (Lampire), 1mM EDTA (Invitrogen) in HBSS without Ca 2+ , Mg 2+ and Phenol Red (Corning)) - 40 mL for blood, 25 mL for spleen, 10 mL for spinal cord. Samples were centrifuged (400 RCF, 5 min, 4°C; settings used for all remaining centrifuge steps), resuspended in HBSS, then centrifuged. Total spleen cell counts were assessed using the EVE Automated Cell Counter (NanoEntek) according to manufacturer’s instructions. Pellets were resuspended in Zombie Red Fixable Viability Dye (1:500 in 100 µL of HBSS, BioLegend) according to manufacturer’s instructions and incubated for 30 min. Cells were washed with NDS-FACS, centrifuged, then transferred to a 96-well plate. Following resuspension in NDS-FACS and centrifugation, cells were blocked with anti-CD16/32 antibody (clone 93, BioLegend, 1:100) for 20 min. Cells were diluted with NDS-FACS buffer and centrifuged, then split between positive staining and isotype controls. Extracellular antibodies were incubated with cells for 30 min: CD45-APC/Cy7 (clone 30-F11, BioLegend, 1:200) for leukocytes, CD11b-PerCP-Cy5.5 (clone M1/70, BioLegend, 1:200) for myeloid cells, Ly6G-Pacific Blue (clone 1A8, BioLegend, 1:200) or its isotype (Rat IgG2a, BioLegend, 1:200) for neutrophils, and Goat anti-Human/Mouse myeloperoxidase/MPO (AF3667, R&D systems, 1:50) or no antibody for myeloperoxidase. Cells were diluted with NDS-FACS buffer and centrifuged twice. For the MPO antibody, all cells were incubated with donkey anti-goat Alexa Fluor 647 (A-21447, Invitrogen, 1:2000) for 30 min, then washed with NDS-FACS buffer and centrifuged twice. Cells were fixed with Cyto-Fast Fix Perm solution (BioLegend) for 20 min, then washed according to manufacturer’s instructions. For overnight storage at 4°C, cells were centrifuged and resuspended in NDS-FACS buffer. The next day, cells were resuspended in Cyto-Fast Perm Wash buffer (BioLegend) and centrifuged. Intracellular staining for citrullinated histones was accomplished with rabbit anti-CitH3 antibody (Abcam, 1:2000 in 100 µL Perm Wash buffer) or a no antibody control incubation for 30 min. Cells were resuspended in NDS-FACS buffer and centrifuged twice. CitH3 was visualized with donkey anti-rabbit Alexa Fluor 488 (Invitrogen, 1:2000 in NDS-FACS buffer), 30 min incubation. Cells were washed with NDS-FACS buffer and resuspended prior to analysis. Cells were assessed with a BD Fortessa X-20 flow cytometer and data were analyzed with FlowJo software. ETs Capture ELISA ET complexes were quantified as previously described 28 . Briefly, a Nunc uncoated 96-well plate was coated with CitH3 capture antibody (Abcam, 1:250) diluted in coating buffer (15mM Na 2 CO 3 , 35mM NaHCO 3 in 1X PBS) and incubated overnight at 4°C. Plates were washed with 1X PBS (x3), then blocked with 5% Bovine Serum Albumin (BSA) for 2 hours at RT. Cell-free supernatant of spinal cord and blood were diluted with 1% BSA in PBS and incubated on plate overnight at 4°C. Following this, the plate was washed (1% BSA, 0.05% Tween-20 in 1X PBS, 3×200 µL) three times, then incubated for 2 hrs at RT with anti-DNA-POD detection antibody (Roche, 1:100) according to manufacturer instructions. Plate was washed with wash buffer three times, then incubated with room temperature TMB substrate solution (1X, Invitrogen) in the dark at RT for about 10 min. The reaction was quenched with 0.5 M phosphoric acid. The absorbance of each well was taken at 450 nm. White Matter Sparing White matter sparing was assessed via Eriochrome cyanine staining as previously described 28 . Tissue sections were cleared in Citrisolv (5 min, Decon Labs), then hydrated in a series of ethanol baths (100% x 2, 95%, 70%, 50%, 1 min) followed by distilled water (2 min). Myelin was stained with Eriochrome Cyanine R solution (0.2% Eriochrome Cyanine R, 90 mM H 2 SO 4 , 5.6% FeCl 3 in dH 2 O) for 10 min followed by three water washes (ddH 2 O, 1 min). Sections were differentiated with 5.6% FeCl 3 in water for 3 min, then washed in ddH 2 O three times. Sections were rehydrated in a series of ethanol baths (70%, 1 min; 95%, 2 min; 100%, 1 min x 2), then cleared with Citrisolv (5 min x 2). Slides were airdried, then coverslipped with Cytoseal. Images were acquired with a 10 X objective on the Leica DM6B upright microscope (RRID: SCR_022128). Tissue sparing was analyzed as previously described using ImageJ—total section area and white matter area were quantified in tissue sections 250 µm apart spanning the lesion site. The section with the lowest percentage of white matter was identified as the lesion center. Single Cell RNA Sequencing Analysis Single-cell RNA sequencing (scRNA-seq) datasets (accessions SRR17591922–SRR17591925) were downloaded from the Sequence Read Archive (SRA) using the NCBI SRA Toolkit (version 3.0). Raw files were converted to FASTQ format, sequencing data were aligned to the mouse transcriptome reference (GRCm39-2024-A), and unique molecular identifiers (UMIs) were quantified using Cell Ranger software (version 9.0.1). The resulting feature-barcode matrices were analyzed using the Seurat package (version 4.3) in R (version 4.2.2). Quality control filters removed cells expressing fewer than 200 or more than 4000 genes, as well as cells with mitochondrial genes above 2%. Cells were clustered using the shared nearest neighbor algorithm and visualized via Uniform Manifold Approximation and Projection (UMAP). Sex classification of neutrophils was based on expression of the female-specific marker Xist and the male-specific marker Ddx3y 38 . Differential expression analysis comparing male and female neutrophils was performed for the Padi gene family (Padi1, Padi2, Padi3, Padi4, and Padi6). Statistics Statistics were analyzed using GraphPad Prism 10. Flow cytometry, ELISA, tissue sparing, and LRWT data were analyzed as follows: for pairwise comparisons, an F test to compare variances was performed, followed by an unpaired two-tailed t-test. If variances were significantly different, Welsh’s correction was used. For comparisons between groups with two or more factors (i.e., genotype and sex), a two-way ANOVA followed by Tukey’s post-hoc test for multiple comparisons was performed. BMS and tissue sparing were analyzed as follows: for comparisons between groups with three or more factors (i.e., treatment, sex, and time), a three-way repeated measures ANOVA or appropriate mixed-effects model was used followed by Tukey’s post-hoc test for multiple comparisons. For scRNAseq, transcript level comparisons were made using the Mann-Whitney U test. Exclusions For data from ELISA and FC experiments, statistical outliers were removed using Grubbs’ test (alpha=0.05). For short term studies, animals with non-standard surgeries (abnormal impact force or displacement) were excluded from analyses. For long-term studies, data from animals that had an abnormal impact force or displacement, died during the study, or exhibited a BMS score > 2 at 1 dpi were excluded from analyses. Excluded data is retained in the data repository associated with this manuscript. Funding This work was supported by funding from The Craig H. Neilsen Foundation Grant #648714, Mission Connect (a program of TIRR Foundation), and NIH R01NS122961. Declaration of Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. CRediT Authorship Statement Shelby Reid: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Visualization, Data Curation, Writing - Original Draft, Project administration. Ashley Tran: Validation, Investigation. Miranda Leal-Garcia : Methodology, Investigation. Sachit Devaraj: Investigation. Mustafa Ozturgut: Data Curation, Formal Analysis, Visualization. Dylan McCreedy: Writing – review & editing, Resources, Supervision, Project administration, Funding acquisition, Conceptualization Data availability All associated data from this manuscript will be deposited in the Open Data Commons for Spinal Cord Injury (ODC-SCI) upon publication ( https://odc-sci.org/ ). Supplemental Figures Download figure Open in new tab Figure S1: PAD4 KO alters neutrophil effector function at 24 hours after SCI. (A) At 24 hr following moderate T9 contusion SCI, Padi4 (-/-) and WT mouse spinal cords and blood were collected and assessed for ETosis via flow cytometry and capture ELISA. (B) Extracellular trap (ET) complexes at the spinal cord injury site were reduced in Padi4(-/-) mice compared to littermate controls (Unpaired Student’s t -test, n=4-5, p=0.0440). (C-D) Intracellular citrullination of histone 3 (CitH3) in (C) blood neutrophils was reduced after injury compared to littermate controls (Unpaired student’s t -test, n=4, p=0.0083). A similar trend was observed in (D) spinal cord neutrophils (Unpaired student’s t -test, n=4, p=0.1507). Data shown as mean ± SEM. *p<0.05, **p<0.01. (E-F) Extracellular myeloperoxidase (MPO) was elevated on (E) blood neutrophils while no change was observed in (F) spinal cord neutrophils. (Unpaired t-test; n=4, p blood =0.0185, p spinalcord = 0.7170). Acknowledgements The authors would like to thank Texas A&M University Microscopy and Imaging Center (RRID: SCR_022128) and Texas A&M University Flow Cytometry & Cell Sorting Facility for the use of their equipment. The diagrams in Figures 1A and S1A were created using Biorender.com. Funder Information Declared Craig H Neilsen Foundation , 648714 National Institute of Neurological Disorders and Stroke , R01NS122961 Footnotes Author Emails Shelby K. Reid: shelbykrr{at}tamu.edu Ashley V. Tran: ashleyvitran{at}tamu.edu Miranda E. Leal: mleal{at}bio.tamu.edu Sachit Devaraj: sachitd97{at}tamu.edu Mustafa Ozturgut: mozturgut{at}tamu.edu References 1. ↵ Hellenbrand DJ , Quinn CM , Piper ZJ , et al. Inflammation after spinal cord injury: a review of the critical timeline of signaling cues and cellular infiltration . J Neuroinflammation 2021 ; 18 ( 1 ): 284 ; doi: 10.1186/s12974-021-02337-2 . 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Share Sex-dependent effects of peptidylarginine deiminases on neutrophil function and long-term outcomes after spinal cord injury Shelby K. Reid , Ashley V. Tran , Miranda E. Leal-Garcia , Sachit Devaraj , Mustafa Ozturgut , Dylan A. McCreedy bioRxiv 2025.05.08.652924; doi: https://doi.org/10.1101/2025.05.08.652924 Share This Article: Copy Citation Tools Sex-dependent effects of peptidylarginine deiminases on neutrophil function and long-term outcomes after spinal cord injury Shelby K. Reid , Ashley V. Tran , Miranda E. Leal-Garcia , Sachit Devaraj , Mustafa Ozturgut , Dylan A. 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