Serum IL-6, IL-10, TNF-α and IFN-γ bio-signature for neonatal sepsis diagnosis and treatment outcome

Serum IL-6, IL-10, TNF-α and IFN-γ bio-signature for neonatal sepsis diagnosis and treatment outcome | medRxiv /* */ /* */ <!-- <!-- /*! * 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-P4HH5NV'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search Serum IL-6, IL-10, TNF-α and IFN-γ bio-signature for neonatal sepsis diagnosis and treatment outcome View ORCID Profile Suraj T , Riya Ahmed , View ORCID Profile Pradeep Debata , Rajni Gaind , GP Kaushal , Renu Gur , Kirti Nirmal , Ravinder Kaur , View ORCID Profile Sushma Nangia , Vivek Kumar , Dipankar Ghosh , Anmol Chandele , Krishnamohan Atmakuri , View ORCID Profile M Jeeva Sankar , View ORCID Profile Ranjan Kumar Nanda doi: https://doi.org/10.1101/2025.02.12.25320841 Suraj T 1 Translational Health Group, International Centre for Genetic Engineering and Biotechnology , New Delhi, India , 110067 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Suraj T Riya Ahmed 1 Translational Health Group, International Centre for Genetic Engineering and Biotechnology , New Delhi, India , 110067 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Pradeep Debata 2 Department of Paediatrics, Vardhman Mahavir Medical College and Safdarjung Hospital India , New Delhi, India , 110029 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Pradeep Debata Rajni Gaind 3 Department of Microbiology, Vardhman Mahavir Medical College and Safdarjung Hospital India , New Delhi, India , 110029 Find this author on Google Scholar Find this author on PubMed Search for this author on this site GP Kaushal 4 Department of Paediatrics, Dr. Baba Saheb Ambedkar Hospital , New Delhi, India , 110085 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Renu Gur 5 Department of Microbiology, Dr. Baba Saheb Ambedkar Hospital , New Delhi, India , 110085 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Kirti Nirmal 6 Department of Microbiology, Guru Teg Bahadur Hospital , New Delhi, India , 110095 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ravinder Kaur 7 Department of Microbiology, Lady Hardinge Medical College and Associated Hospitals , New Delhi, India , 110001 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Sushma Nangia 8 Department of Neonatology, Lady Hardinge Medical College and Associated Hospitals , New Delhi, India , 110001 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Sushma Nangia Vivek Kumar 9 Department of Paediatrics, All India Institute of Medical Sciences , New Delhi, India , 110029 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Dipankar Ghosh 10 Special Centre for Molecular Medicine, Jawaharlal Nehru University , New Delhi, India , 110067 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Anmol Chandele 11 ICGEB-Emory Vaccine Centre, International Centre for Genetic Engineering and Biotechnology , New Delhi, India , 110067 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Krishnamohan Atmakuri 12 Bacterial Pathogenesis Group, Centre for Microbial Research Translational Health Science and Technology Institute , Faridabad, Haryana, India , 121004 Find this author on Google Scholar Find this author on PubMed Search for this author on this site M Jeeva Sankar 9 Department of Paediatrics, All India Institute of Medical Sciences , New Delhi, India , 110029 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for M Jeeva Sankar For correspondence: ranjan{at}icgeb.res.in jeevasankar{at}gmail.com Ranjan Kumar Nanda 1 Translational Health Group, International Centre for Genetic Engineering and Biotechnology , New Delhi, India , 110067 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Ranjan Kumar Nanda For correspondence: ranjan{at}icgeb.res.in jeevasankar{at}gmail.com Abstract Full Text Info/History Metrics Supplementary material Data/Code Preview PDF Abstract Background Diagnosing sepsis in preterm neonates is a significant challenge, underscoring the urgent need for timely and accurate methods. Serum inflammatory protein signatures show promising potential for early and precise disease diagnosis. Methods In this study, a cohort of preterm neonates (n=50, 25-35 weeks gestation, female 40%) at the time of suspicion and follow-up were enrolled along with healthy neonates. Based on clinical presentation and blood culture or MALDI results, the sepsis suspicion cases were categorized into culture-positive (CP)/culture-negative (CN) sepsis (n=13 each) or no-sepsis (NS, n=12) and compared with healthy controls (HC, n=12) from similar settings. These sub-groups (CP, CN, and NS) were followed up till completion of antibiotic therapy. Serum inflammatory proteins and trace elementals ( 57 Fe, 66 Zn, 63 Cu, 77 Se, 44 Ca, 24 Mg) were profiled using the Olink® Target 96 inflammation panel and inductively coupled plasma mass spectrometry (ICP-MS), respectively. Serum proteins with log 2 fold changes>±1.3 and p<0.05 were identified as differentially expressed proteins (DEPs) and monitored in the follow-up samples receiving treatment. Elements showing significant differences (p<0.05) between the study groups were also identified for correlation analysis. Results Serum inflammatory protein levels showed group-specific trends. Significantly higher serum IL-6, IL-10, TNF-α and LIF levels with low IFN-γ and CCL28 were observed in the CP sepsis group compared to HC. CN group also showed reduced serum IFN-γ along with CXCL10 and CCL11 levels when compared to HC. Also, the serum IL-6 level in CP cases was positively correlated with IFN-γ and IL-10 levels (r<0.77, p<0.05), and an increased IL6:IFN-γ was observed in the CP, CN and NS groups compared to the HC. Serum of NS patients showed higher FGF-23 levels with lower IFN-γ and CCL11 than HC. Upon recovery, the serum IL-6 levels reached normal levels in CP, whereas the CN group showed IFN-γ, CXCL10, and CCL11 levels returned to normal. Serum iron levels were significantly reduced in both the CP and CN groups, while serum selenium was low in the CP group and serum copper was lower in the CN group, with all levels returning to normal upon recovery. Conclusions Absolute levels of IL-6, IL-10, IFN-γ, and TNF-α in serum can be used as biosignatures for neonatal sepsis, offering the potential for early disease diagnosis and monitoring therapeutic response. Additionally, these markers implicated in disease resolution mechanisms might serve as therapeutic targets in sepsis treatment. Introduction Neonatal sepsis is a severe and complex systemic response to infections caused by bacteria, viruses, or fungi, leading to significant morbidity and mortality. It is traditionally classified into early-onset sepsis (EOS), which occurs within 72 hours of birth, and late-onset sepsis (LOS), which appears after 72 hours. The clinical presentation is often non-specific, with symptoms including temperature instability, respiratory distress, feed intolerance, lethargy, and, in severe cases, multi-organ dysfunction ( 1 ). Accurate and timely diagnosis of neonatal sepsis remains a significant challenge due to its non-specific presentation and overlap with non-infectious conditions. Current diagnostic methods primarily rely on laboratory-based microbiology culture techniques for pathogen identification, supplemented by advanced tools such as MALDI-TOF mass spectrometry and multiplex PCR. Other diagnostic approaches include assessing multiple markers, such as inflammatory proteins, micronutrient analysis, white blood cell counts, and cytokine analysis. However, accurate and early diagnosis requires multi-dimensional strategies integrating clinical symptoms with biomarkers ( 2 , 3 ). Inflammation is pivotal in neonatal sepsis, representing a complex immunological response beyond mere pathogen presence. In newborns, the immature immune system exaggerates inflammatory response, potentially causing more harm than the initial infection. The inflammatory cascade in neonatal sepsis involves the release of various mediators, including pro-inflammatory cytokines (IL-1β, IL-8, IL-6, TNF-α), acute phase proteins (C-reactive protein, procalcitonin), and damage-associated molecular patterns (DAMPs). Traditional diagnostic approaches rely heavily on blood culture results, which take 24-48 hours and may yield false negatives in cases of prior antibiotic exposure. Within hours of infection, the inflammatory response begins, and identification of the inflammatory markers may provide a more comprehensive approach to sepsis diagnosis ( 4 , 5 ). Dysregulation in serum micronutrients also plays a crucial role in the pathophysiology and progression of neonatal sepsis. Zinc deficiency, commonly observed in septic neonates, compromises immune function by affecting T-cell development, cytokine production, and neutrophil activity. Selenium, an essential trace element with antioxidant properties, demonstrates notably reduced levels during septic episodes, potentially contributing to increased oxidative stress and cellular damage. Iron metabolism undergoes significant alterations during sepsis, with iron deficiency and overload potentially compromising immune response and increasing susceptibility to infection ( 6 ). Systematic profiling of inflammatory markers, such as cytokines, chemokines, and other immune mediators, will not only provide details of neonatal sepsis pathophysiology but also may help in differentiate various sepsis subtypes. Early, preferably on-time differentiation will equip clinicians to initiate targeted interventions before getting the microbiological results, thereby improving patient outcomes in this vulnerable population where time to appropriate treatment is crucial. Similarly, micronutrient profiling may provide valuable insights into disease phenotype and offer potential supplementation strategies as therapeutic targets. In this study, we analysed serum inflammatory proteins and micronutrient profiles in neonate with sepsis during diagnosis and after treatment completion and in healthy controls. The identified molecular signatures shed light on sepsis presentation but also hold potential for developing improved diagnostics for timely and accurate classification and appropriate therapeutic interventions. Materials and methods Ethics committee approval This multi-centre study was approved by the ethics committees of the Vardhman Mahavir Medical College and Safdarjung Hospital (IEC/VMMC/SJH/Project/2021-05/CC-157, dated 24.07.2021), All India Institute of Medical Sciences (IEC-1074/06.11.20, RP-28/2020), Dr. Baba Saheb Ambedkar Medical College and Hospital (5(32)/2020/BSAH/DNB/PF, dated 31.08.2020), Lady Hardinge Medical College (LHMC/IEC/2022/03/30), University College of Medical Sciences & Guru Teg Bahadur Hospital (IECHC-2022-51-R1, dated 07.12.2022), and the International Centre for Genetic Engineering and Biotechnology (ICGEB/IEC/2021/28, version 2). Subject recruitment and classification In this prospective cohort study, we enrolled preterm neonates born between 25 and 35 weeks of gestation who were admitted to the neonatal intensive care units (NICU) of the participating hospitals over a period of 18 months after obtaining informed written consent from their legal guardians. Neonates with major congenital malformations or those born to mothers with HIV or hepatitis B, as well as those with postnatal age exceeding 28 days, were excluded from the study. Neonates enrolled in the study were suspected of sepsis based on maternal/perinatal risk factors or clinical signs. Upon suspicion, a diagnostic work-up, including a sepsis screen and blood culture, was performed before initiating antibiotics. The sepsis screen assessed four parameters: low absolute neutrophil count (0.2), CRP (>6 mg/L), and or erythrocyte sedimentation rate (ESR)>15 mm in the first hour, with any two abnormalities confirming a positive result. Neonates were classified into four groups: culture-positive (CP) sepsis, culture-negative (CN) sepsis, no sepsis (NS), and healthy controls (HC). CP cases had true pathogens in blood cultures, with coagulase-negative Staphylococcus classified as CP only if sepsis symptoms and antibiotic treatment for ≥5 days supported the diagnosis. CN sepsis was identified when cultures were sterile or showed commensals, but sepsis screen results or clinical course indicated sepsis. NS cases did not meet CP or CN criteria. Antibiotic decisions followed unit-specific protocols. A set of neonates (n=38, CP/CN/NS: 13/13/12) were followed up till recovery (details presented in Table S1). Clinically stable neonates with no symptoms of sepsis within the first 5 days of life were enrolled as healthy controls (HC, n=12). Serum sample preparation At the time of diagnosis or follow-up, whole blood was collected from the neonates using BD Vacutainer® blood collection tubes and an aliquot of it was loaded into the automated culture bottles (BD BACTEC or BACT/ALERT) tube. The other aliquot was incubated for 10-30 minutes at room temperature and centrifuged at 1,000 g and 10 minutes to harvest serum samples. These serum aliquots were transported and stored at -80° C till further processing. Serum proteomic profiling Serum cytokines, and chemokines of neonates with sepsis (n=38; CP/CN/NS: 13/13/12) at the time of suspicion and again after treatment completion and healthy controls (HC; n=12) were profiled using a Proximity Extension Assay (Olink® Target 96 Inflammation panel, Olink Proteomics AB, Salagatan 16F, SE-753 30 Uppsala, Sweden). Briefly, this technology utilizes a pair of antibodies labeled with unique DNA oligonucleotides, which bind to the target proteins. When these antibodies come into close proximity, their oligonucleotides hybridize, allowing DNA polymerase to extend the hybridized sequences and create unique PCR-amplifiable DNA reporter sequences for each detected protein. These reporter sequences are then amplified via PCR, quantified through high-throughput real-time PCR, and normalized using internal controls (Incubation control, Extension control and the Detection control) to generate Normalized Protein eXpression (NPX) values on a log 2 scale. The higher NPX values suggest a higher protein expression. Detection panels are subjected to rigorous internal and external quality control (QC) procedures to ensure data accuracy and reliability. Internally, the QC process incorporates four distinct control measures: two immunoassay controls, one extension control, and one detection control. Externally, eight standardized samples are utilized to evaluate assay performance. These samples serve multiple critical functions, including the calculation of within-batch and between-batch coefficients of variation, determination of the limit of detection, and facilitation of data normalization. Samples have passed QC when their NPX values deviate by less than 0.3 from the plate’s median NPX value. Scatterplots are generated to visualize the distribution of NPX values across all samples to identify potential outliers within the dataset, enabling the detection of any anomalies or deviations from expected patterns. GO enrichment analysis and pathway enrichment analysis The deregulated protein sets were selected for GO enrichment and pathway enrichment analysis employing ShinyGO 0.81( https://bioinformatics.sdstate.edu , Ge SX, Jung D & Yao R, Bioinformatics 36:2628– 2629, 2020) and the top 20 GO terms and KEGG pathways were taken. Serum micronutrient analysis using Inductively Coupled Plasma Mass Spectrometry (ICP-MS) Due to limited sample availability a subset of the study (CP/CN/HC: 5/7/7) was used for serum micronutrients ( 57 Fe, 66 Zn, 63 Cu, 77 Se, 44 Ca, 24 Mg) profiling using ICP-MS (Thermo Scientific iCAP-TQ mass spectrometer). Briefly, serum samples (50 µL) were digested using 75 ml nitric acid (70%, 225711, Sigma, USA and 25ml hydrogen peroxide (Supelco, 107298, Hydrogen peroxide 30% Suprapur®) in a microwave digester (Anton Paar, USA) at 140°C for 30 minutes. After cooling, the diluted serum samples were prepared with ultrapure water (Honeywell water) to reduce the acid concentration to the permissible limit to inject into ICP-MS. The diluted acid-digested serum samples with the appropriate standard elemental mixture (92091, Sigma Aldrich, 0.1-1000 ppm) were used for trace element level quantification using Kinetic Energy Discrimination (KED) mode, using helium as a carrier gas. The data were analyzed using Qtegra (Thermo Scientific) software, and the final concentration of the elements was calculated using the standard plot. Statistical analysis Statistical significance between groups was performed using univariate statistical tools like Welch’s t-test, parametric (unpaired or paired) and a p-value <0.05 was considered statistically significant. The proteins with a p-value ±1.3 were selected as differentially expressed proteins (DEPs). Spearman (two-tailed) correlation analysis between the expression levels of DEPs, and trace elements was carried out using the GraphPad Prism version 8.4.2 for Windows (GraphPad Software, Boston, Massachusetts USA, www.graphpad.com ). If the value is close to ±1.0 was considered as the strongest correlation. Also, the Spearman correlation (two-tailed) value was calculated between IL6 and IFN-γ, IL-10. Results Demographic details of the study subjects In this prospective observational study, 88 samples belonging to different sepsis groups (CP/CN: 13/13) and control groups (NS/HC, 12/12) were used. Females consisted of 40% of each study group. Acinetobacter baumannii , coagulase-negative Staphylococcus (CoNS) and Klebsiella pneumonia were isolated in neonates with CP sepsis. Most CP and CN sepsis neonates had early-onset sepsis (EOS) cases. Serum inflammation protein signature showed group-specific variations in neonatal sepsis Out of the 92 proteins (details in Table S2) monitored in all study groups, 89 passed the QC (as described in the method section). Three proteins (Beta-NGF, IL-24, and Thymic stromal lymphopoietin (TSLP)) had a coefficient of variation (CV) of more than 25 % of CV and were excluded from the study. A few serum samples (CP, n=4; CN, n=2; NS, n=3; HC, n=1) did not pass the QC (as described in the method section), and were excluded. Between the CP and CN vs HC, a set of 4 and 3 DEPs (log 2 FC>±1.3, p-value < 0.05), respectively, were identified ( Figure 1B, C ). Compared to HC, significantly high serum IL-10, IL-6, LIF, and TNF-α levels and low CCL28 and IFN-γ levels were observed in the CP group ( Figure 1B ), serum IFN-γ, CXCL10, and CCL11 levels were significantly lower in the CN group ( Figure 1C ). Between CP and CN, a significantly higher abundance (log 2 FC>±1.3, p-value ±1.3, p-value ±1.3, p-value ±1.3, p-value < 0.05) were identified between NS and HC groups. Serum FGF-23 levels in the NS group were significantly high, and IFN-γ and CCL11 were low (Supplementary Figure S1C). Out of these 12 DEPs, at least between two groups, a set of five molecules (IL-6, LIF, IFN-γ, CCL11, FGF-23) showed significant deregulation between sepsis suspects and HC ( Figure 1E ). Download figure Open in new tab Figure 1. Serum inflammatory proteins show differential expression in neonatal sepsis cases compared to the healthy control (HC). A. Schematic diagram representing the workflow used for OLink Proteomics analysis. B . Volcano plots showing dysregulated proteins (log 2 FC>±1.3, p-value < 0.05) between study groups {Culture Positive (CP) and Healthy control (HC), Culture Negative (CN) and HC, CP and CN. C. Box plots showing the abundances of dysregulated analytes between sepsis suspects and healthy controls (HC) and their group-specific changes between CP, CN and NS. Serum micronutrients of neonatal sepsis patients showed variations Out of the six ( 57 Fe, 66 Zn, 63 Cu, 77 Se, 44 Ca, 24 Mg) serum elemental levels monitored, iron showed significant reduction (p<0.05) in both CP and CN sepsis subjects compared to HC ( Figure 2A ). CP patients had significantly lower serum selenium levels than HC ( Figure 2B ). Additionally, copper levels in the CN groups were significantly (p<0.05) low compared to the HC ( Figure 2B ). The rest of the elements were similar between the study groups. In the recovered CP subjects, selenium levels recovered while serum iron levels were similar to the initial levels observed at the time of diagnosis. The serum Fe and Cu levels in the CN groups completing therapy recovered to normal levels ( Figure 2C ). Download figure Open in new tab Figure 2: Serum elemental composition of sepsis patients showed variations. A. Schematic diagram representing the workflow used for ICP-MS. B. Box plot of serum Iron, Copper, Calcium, Magnesium, Zinc, and Selenium between CP, CN and HC. C. Box plots depicting the serum Iron, Selenium, and Copper levels in followed-up neonatal sepsis cases upon recovery compared to HC. Protein-protein interaction analysis of the deregulated serum proteins and elements showed dysregulated TNF, JAK-STAT, and NF-Kappa B pathways between sepsis sub-groups The DEPs were selected for the protein-protein interaction (PPI) analysis using the Cytoscape tool ( Figure 3A ). The identified perturbed pathways, with FDR< 0.05, between CP and HC) were TNF, JAK-STAT, and T-cell receptor signaling pathways ( Figure 3B ). Further in the CN, the IL-17, Th17, TNF signaling pathway and chemokine signalling pathways were dysregulated ( Figure 3B ). Between the CP and CN, TLR, chemokine, IL-17, TNF, and NF-Kappa B signaling pathways were perturbed ( Figure 3B ). A positive correlation was observed between IL-6 and IFN-γ in the CP group only ( Figure 3C ). Between serum IL-6 and IL-10, a significant positive correlation was observed in the CP and CN sepsis groups ( Figure 3C ). A significantly higher IL-6/ IFN-γ ratio was observed in the sepsis suspects groups (CP, CN and NS) compared to HC ( Figure 3D ). Download figure Open in new tab Figure 3: Protein-Protein interaction, Pathway analysis and correlation analysis. A. The protein-protein interaction network of the sorted DEPs between the different sub-groups of neonatal sepsis was constructed using Cytoscape software. B. Bar plot depicting the significantly dysregulated pathways between the Sub-groups. C. Correlation heat map (Pearson r values) of inflammatory proteins and microelements. Red indicates a positive correlation, and blue shows a negative correlation. Correlation (Spearman r) between IL-6 and IFN-γ, IL-6 and IL-10. D. Box plots with IL-6/IFN-γ ratios and IL-6/IL-10 ratios between the sub-groups. Serum inflammation markers in neonatal sepsis cases receiving treatment show specific variations The sepsis neonates were followed up till they completed antibiotic therapy and recovered from the clinical symptoms. IL-6 significantly recovered and came to HC level, while the rest remained similar ( Figure 4A ). On completing treatment, the serum IFN-γ, CXCL10 and CCL11 levels of CN groups, showed significant variations and were closer to the HC group ( Figure 4B ). In recovered NS patients, serum FGF-23 and CCL11 levels showed significant changes (p<0.05), returning to levels similar to HC, while the other two dysregulated proteins remained unchanged. ( Figure 4C ). Download figure Open in new tab Figure 4: Box plots showing the trend of important serum proteins in the follow follow-up culture, positive, culture negative, and no sepsis groups. Abundance of important serum protein at diagnosis and recovery in culture positive (A: CP), culture negative (B: CN) and no sepsis groups (C: NS). Download figure Open in new tab Figure 5: Schematics representing pathophysiological changes in A . culture positive (CP) and B. culture negative (CN) neonatal sepsis cases. Discussion Neonatal sepsis represents a potentially devastating systemic infection, which can be mono or polymicrobial in origin, and it poses a significant threat to newborns due to their immature immune systems and nonspecific clinical presentations. This condition remains a leading cause of neonatal mortality worldwide, particularly in developing countries, emphasizing the critical importance of early recognition and prompt therapeutic intervention ( 7 ). Based on the blood culture and mass spectrometry results, neonatal sepsis patients were grouped into culture positive (CP), Culture negative (CN) and No sepsis (NS). The CP sepsis group showed a distinct inflammatory signature characterized by elevated levels of pro-inflammatory cytokines IL-6, IL-10 LIF, and TNF-α, decreased IFN-γ, and CCL28. IL-6, is a sentinel cytokine produced by various cell types, including endothelial cells, fibroblasts, mononuclear phagocytes and placental tissues (amnion, decidua, trophoblast and chorion), exhibits rapid surge following microbial stimulation ( 8 , 9 ). In the inflammatory cascade, IL-6 upregulation precedes C-reactive protein synthesis and leads to subsequent release of TNF-α, thus serving as an important early marker of infection. IL-6 plays a pivotal role in immune activation through multiple mechanisms, including B-cell antibody secretion, T-cell activation, especially the cytotoxic T-cell and the orchestration of downstream cytokine release, particularly TNF-α and IL-1β. As an early diagnostic marker of neonatal sepsis, IL-6 demonstrates a characteristic elevation several hours before C-reactive protein increase, and when these markers are evaluated in combination, their diagnostic sensitivity approaches 100%, underscoring their critical importance in clinical assessment ( 10 ). Elevated serum IL-6 was observed in severe and moderate COVID-19 patients compared to the mild cases, indicating its importance as a marker of hyperinflammation and a strong predictor of mortality in COVID-19( 11 ). IL-6 is also reported as a disease progression marker in Rheumatoid Arthritis (RA) ( 12 ). Recent studies have also reported IL-6 levels to be high in neonates with sepsis and their levels significantly getting reduced upon treatment ( 13 ). In our study, higher serum IL-6 levels were observed in the CP neonates at the time of suspicion and upon recovery, these levels are getting similar to HC. Among immune cells, Monocytes and Macrophages are the primary sources of TNF-α ( 14 ). Clinical studies have shown that with onset of bacterial infection, TNF-α levels become detectable in blood ( 15 ). TNF-α along with IL-1 are one of the few inflammatory mediators often reported at inflammation sites during initial phase ( 16 ). In neonatal sepsis, TNF-α causes increased vasodilation, permeability which leads to systemic edema ultimately causing septic shock. Especially in early-onset neonatal cases, these pathological changes can rapidly progress to multiple-organ failure and death ( 17 ). Consistent with these findings, we also noted that the CP sepsis groups exhibited a similar trend, characterized by elevated TNF-α levels at the time of suspicion. The IL-6 cytokine family encompasses a diverse array of pleiotropic inflammatory mediators, including IL-6, IL-27, IL-31, IL-11, cardiotrophin-1 (CT-1), leukaemia inhibitory factor (LIF), oncostatin M (OSM), ciliary neurotrophic factor (CNTF) and cardiotrophin-like cytokine factor 1 (CLCF1) ( 18 , 19 ). Extensive immunological studies focusing on IL-6, LIF, OSM and IL-27 have highlighted their key roles in host defence mechanisms, demonstrating pivotal contributions to antimicrobial and antiviral immunity by providing essential tissue protection against infection-induced pathological injury ( 20 ). So, higher serum IL-6 and LIF levels were observed in the CP group compared to the HC group, which explains that the host immune system is taking care of the infection. IFN-γ plays a critical immune-regulatory role in sepsis be activating monocytes and macrophages, which is necessary for microbial elimination during sepsis by the host. Activated CD4+ and CD8+ T cells and NK cells, are major producers of IFN-γ which are again involved in the upregulating of the expression of HLA-DR expression, thus enhancing antigen presentation and immune response capabilities ( 21 ). Both pre-clinical and clinical sepsis studies show a significant reduction in T cell IFN-γ production, with therapeutic interventions to restore this cytokine to improve survival in experimental models ( 22 ). The critical immune-regulatory role of IFN-γ is further emphasized by observations in infants with deficient production, characterized by compromised neutrophil mobility, reduced NK cell activity, and an increased susceptibility to recurrent infections ( 23 ). Another reason for reduced levels of IFN-γ levels could be due to the migration and accumulation of IFN-γ producing cells at the sites of infection, contributing to its lower circulatory levels. Furthermore, patients with severe sepsis exhibited reduced IFN-γ production, and an imbalance in T helper cell cytokines, characterized by elevated Th2 cytokines and decreased Th1 cytokines ( 24 ). In this study, similar observation in both CP and CN sepsis groups were witnessed, marked by the lower IFN-γ level at diagnosis which strongly suggesting a dysregulated immune response contributing to sepsis – associated immunosuppression. The IFN-γ levels partially getting resolved upon treatment completion in recovered neonates, indicated an improvement of the immune response. IL-6 is reported as a pleiotropic cytokine that can mediate both pro- and anti-inflammatory responses depending on the context of the physiological environment ( 25 ). In contrast, IFN-γ, a type II interferon, enhances antigen presentation through activation of antigen-presenting cells (APCs) and promotes cell-mediated immunity ( 26 ). The IL-6:IFN-γ ratio is critical, which helps determine the direction of the immune response. An elevated IL-6/IFN-γ ratio, as observed in infectious diseases, potentially drives exuberant inflammation and subsequent damage to tissues ( 27 ). In this study, a positive correlation (r=0.77, p<0.05) between IL-6 and IFN-γ was observed in CP sepsis, and a significant increase in the IL6/IFN-γ ratio was observed in the sepsis suspects than HC. Anti-inflammatory cytokines like IL-10 and TGF-β are key modulators of immune homeostasis during sepsis by limiting excessive pro-inflammatory responses. These cytokines act as immunological brakes, preventing the hyper-inflammatory state that could lead to tissue damage and organ dysfunction ( 28 ). Various immune cells, encompassing T and B lymphocytes, monocytes, NK cells and macrophages, synthesize and release IL-10 ( 29 ). This cytokine suppresses the production of pro-inflammatory mediators, including IL-1, IL-6, GM-CSF and TNF-α in cells of the immune system ( 30 ). In this study, we observed significantly elevated IL-10 concentrations CP group, which corresponded with decreased levels of IFN-γ. Interestingly, the level of other Pro-Inflammatory cytokines like IL-6, and TNF-α remains higher in the CP group as the maximum cases in our study group are from the EOS in which the pro-inflammatory effect will dominate the anti-inflammatory one. Studies have revealed a positive monotonic relationship between IL-6 and IL-10 in sepsis patients infected with Gram-positive and Gram-negative bacteria. While this correlation was significant, it was particularly pronounced in sepsis patients infected with Gram-negative bacteria, demonstrating a stronger linear association between these two cytokines ( 31 ). In our study, most of the CP patients were reported with Gram-negative pathogens, and a significant positive correlation between IL-6 and IL-10 was observed in the CP cases (r=0.7473, p<0.05). Interestingly, a positive correction between serum IL-6 and IL-10 levels was also observed in the CN patients (r=0.7692, p<0.05). CCL20 is an important chemokine that plays a crucial role in inflammatory responses as it mainly recruits CCR6-expressing cells, including memory T-cells, B cells, Th17 cells and immature dendritic cells ( 32 ). IL-6 can induce CCL20 production in various cell types as they create a pro-inflammatory environment. Both are involved in Th17 cell differentiation and recruitment. In chronic inflammation, IL-6 and CCL20 form a positive feedback loop where IL-6 induces CCL20 production, which recruits more inflammatory cells that produce IL-6 ( 33 ). TNF-α is a potent inducer of CCL20 expression. They synergistically enhance inflammatory responses. TNF-α can upregulate CCL20 through NF-κB signalling pathways ( 34 ). A high serum CCL20 levels observed in the CP patients corroborates earlier reports ( 13 ). IFN-γ-inducible protein 10 (IP-10/CXCL10), a pivotal chemokine secreted by cells stimulated with type I and II interferons and lipopolysaccharide, acts as an important chemoattractant for activated T lymphocytes ( 35 ). This molecular mediator demonstrates prominent expression in Th1-mediated inflammatory pathologies, playing a fundamental role in orchestrating activated T-cell recruitment to inflammatory tissue microenvironments ( 36 ). This complex immunological relationship between IFN-γ and CXCL10 is also observed in our data by the robust linear relation, wherein diminished IFN-γ levels directly correspond to decreased CXCL10 concentrations, and subsequent immunological recovery is marked by a simultaneous elevation of both cytokine and chemokine levels. Interestingly, low serum IFN-γ, CXCL10 was reported in the neonates suffering from inflammatory diseases like placental histological chorioamnionitis (HCA) using Olink analysis ( 37 ). Overall, the downregulation of CXCL10 and IFN-γ shows overall repressed Th1 immunity. We also observed that iron levels were much lower in sepsis confirmed cases in comparison to HC. This dysregulation aligns to our observation of increases IL-6 levels in sepsis as it known to have role in modulating iron homeostasis ( 38 ). IL-6 can stimulate hepatocytes to secrete various acute-phase proteins, including hepcidin (iron regulatory hormone), CRP, fibrinogen, complement C3, thrombopoietin, and ferritin ( 39 ). Briefly, during inflammation, the IL-6 level increases, which induces the release of hepcidin from hepatocytes through the JAK/STAT3 pathway. The released hepcidin binds to the iron exporter of the cell, called as ferroportin and causes its internalization, ubiquitination and lysosomal degradation. As a result, the intracellular iron concentration increased significantly and to sequester this iron, the ferritin level increases in the cell ( 40 ) which leads to a decrease in the serum iron levels, as observed in the CP and CN sepsis groups. Selenium (Se) is an essential trace element in biological processes mainly regulating selenoprotein synthesis, an important protein known for its antioxidant and anti-inflammatory properties ( 41 ). Se deficiency is often observed in infection. There have been studies indicating that there is reduced Se in sepsis-affected neonates. Se deficiency greatly impairs innate and adaptive immunity by reducing NK cell activity, T cell function and immunoglobin (IgM and IgG) while increasing reactive oxygen species (ROS). This compromised immune function, combined with diminished neutrophil and macrophage activity, increases susceptibility to infections ( 42 ). Elevated serum IL-6 and TNF-α levels observed in the sepsis suspected cases control the Se levels by influencing its bioavailability. Adequate Se levels are essential to promote the expression of selenoproteins, IFN-γ, ( 43 ) which could be contributing to low IFN-γ levels in sepsis suspected cases. Our study also showed that serum selenium levels were low at suspicion, which upon recovery reached to normal levels, indicating the antioxidant attributes of selenium and its possible influence in diminishing mortality related to sepsis in neonates. Elevated copper levels, especially in macrophages during infection, serve as an antimicrobial defence mechanism due to its toxic effects on the pathogens ( 44 ). The copper accumulation observed during fungal infections is primarily driven by the host immune system as an antimicrobial strategy. When copper levels are elevated by the host, it creates oxidative stress conditions around the pathogen; it can disrupt cell membranes, interfere with essential proteins and enzymes and lead to the generation of harmful ROS ( 45 ). This clearly explains the low levels of copper in the serum in the case of the CN group. There is also a significant increase in the copper levels in the serum of subjects completing antibiotic therapy to healthy levels. There are a few limitations of this study. The serum protein abundances were monitored at the time of diagnosis and at completing therapeutic intervention; however, to capture the dynamics of the inflammatory response in neonatal sepsis, multiple time point sampling will be useful. Validation in an independent cohort may be useful to discover the translation potential of the adopted tool and the identified marker signatures. Conclusion Sepsis is a leading cause of morbidity and mortality in neonates and identification of the markers contributing to their immune dysregulation might be useful for early disease diagnosis. This study demonstrates an inflammatory signatures of neonatal sepsis characterized by increased serum pro-inflammatory cytokines such as IL-6, LIF, and TNF-α levels and a decreased IFN-γ and CCL28 levels which are linked to the serum iron, selenium and copper levels. In addition to the commonly used serum IL-6 as marker for sepsis diagnosis, other cytokines also contribute as the central mediator of the inflammatory cascade and a possible recovery marker. This study identified a putative serum biosignature explaining the inflammatory landscape in neonatal sepsis, paving the way for better diagnostic and therapeutic strategies. Data Availability All data produced in the present study are available upon reasonable request to the author Availability of data and materials The data that support the findings of this study are available on request. Competing interests The authors declare that they have no competing interests. Funding The project was funded by the Department of Biotechnology (DBT), India (BT/PR38173/MED/97/474/2020 dated 11.03.2021). Author contributions Conceptualization: R.N. Experiment design: R.N., S.T. Methodology: S.T., R.A. Provided clinical samples and data: P.D., R.G., G.P.K., R.G., K.N., R.K., S.N., V.K. Sample storage and record keeping: R.A., S.T. Clinical data management and analysis: R.A., S.T. Sample processing: S.T. Data acquisition: S.T. Data analysis: S.T. Investigation: S.T. Supervision: R.N., M.J.S Funding acquisition: R.N., R.G., R.K., S.N., G.P.K., R.G., K.N., M.J.S. Coordination and strategy: R.N., M.J.S. Writing original draft: S.T. and R.N. Writing-review and editing: S.T., R.N., R.G., R.K., S.N., G.P.K., R.G., K.N.,V.K., D.K., A.C., K.A., M.J.S. All authors read and approved the final manuscript and had full access to all the data in the study. Acknowledgements We thank the hospital staff of the clinical sites for helping us with sample collection and pre-processing. We would like to acknowledge Dr(s) Sushil Shrivastava, Meetu Salhan, Harsh Chellani, Pratima Anand, and Narendra Pal Singh for their constant support and involvement in this project. We would also like to thank the Department of Biotechnology (DBT), India, for financial support. 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Share Serum IL-6, IL-10, TNF-α and IFN-γ bio-signature for neonatal sepsis diagnosis and treatment outcome Suraj T , Riya Ahmed , Pradeep Debata , Rajni Gaind , GP Kaushal , Renu Gur , Kirti Nirmal , Ravinder Kaur , Sushma Nangia , Vivek Kumar , Dipankar Ghosh , Anmol Chandele , Krishnamohan Atmakuri , M Jeeva Sankar , Ranjan Kumar Nanda medRxiv 2025.02.12.25320841; doi: https://doi.org/10.1101/2025.02.12.25320841 Share This Article: Copy Citation Tools Serum IL-6, IL-10, TNF-α and IFN-γ bio-signature for neonatal sepsis diagnosis and treatment outcome Suraj T , Riya Ahmed , Pradeep Debata , Rajni Gaind , GP Kaushal , Renu Gur , Kirti Nirmal , Ravinder Kaur , Sushma Nangia , Vivek Kumar , Dipankar Ghosh , Anmol Chandele , Krishnamohan Atmakuri , M Jeeva Sankar , Ranjan Kumar Nanda medRxiv 2025.02.12.25320841; doi: https://doi.org/10.1101/2025.02.12.25320841 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Pediatrics Subject Areas All Articles Addiction Medicine (568) Allergy and Immunology (863) Anesthesia (297) Cardiovascular Medicine (4421) Dentistry and Oral Medicine (443) Dermatology (382) Emergency Medicine (606) Endocrinology (including Diabetes Mellitus and Metabolic Disease) (1507) Epidemiology (15212) Forensic Medicine (30) Gastroenterology (1121) Genetic and Genomic Medicine (6581) Geriatric Medicine (667) Health Economics (996) Health Informatics (4520) Health Policy (1366) Health Systems and Quality Improvement (1611) Hematology (539) HIV/AIDS (1264) Infectious Diseases (except HIV/AIDS) (15906) Intensive Care and Critical Care Medicine (1103) Medical Education (620) Medical Ethics (144) Nephrology (667) Neurology (6580) Nursing (345) Nutrition (998) Obstetrics and Gynecology (1141) Occupational and Environmental Health (956) Oncology (3324) Ophthalmology (970) Orthopedics (369) Otolaryngology (420) Pain Medicine (435) Palliative Medicine (129) Pathology (663) Pediatrics (1689) Pharmacology and Therapeutics (691) Primary Care Research (710) Psychiatry and Clinical Psychology (5432) Public and Global Health (9212) Radiology and Imaging (2193) Rehabilitation Medicine and Physical Therapy (1368) Respiratory Medicine (1194) Rheumatology (593) Sexual and Reproductive Health (709) Sports Medicine (529) Surgery (709) Toxicology (99) Transplantation (288) Urology (265) (function(){function c(){var b=a.contentDocument||a.contentWindow.document;if(b){var d=b.createElement('script');d.innerHTML="window.__CF$cv$params={r:'9ff4456389340708',t:'MTc3OTM3Mzc0Mg=='};var a=document.createElement('script');a.src='/cdn-cgi/challenge-platform/scripts/jsd/main.js';document.getElementsByTagName('head')[0].appendChild(a);";b.getElementsByTagName('head')[0].appendChild(d)}}if(document.body){var a=document.createElement('iframe');a.height=1;a.width=1;a.style.position='absolute';a.style.top=0;a.style.left=0;a.style.border='none';a.style.visibility='hidden';document.body.appendChild(a);if('loading'!==document.readyState)c();else if(window.addEventListener)document.addEventListener('DOMContentLoaded',c);else{var e=document.onreadystatechange||function(){};document.onreadystatechange=function(b){e(b);'loading'!==document.readyState&&(document.onreadystatechange=e,c())}}}})();