New Insight Into the Neuroimmune Interplay In Pseudomonas aeruginosa Keratitis

New Insight Into the Neuroimmune Interplay In Pseudomonas aeruginosa Keratitis | 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 New Insight Into the Neuroimmune Interplay In Pseudomonas aeruginosa Keratitis Naman Gupta , Giovanni LoGrasso , Linda D. Hazlett , View ORCID Profile Shunbin Xu doi: https://doi.org/10.1101/2025.03.06.641908 Naman Gupta Department of Ophthalmology, Visual and Anatomical Sciences, Wayne State University School of Medicine/Kresge Eye Institute , Detroit, Michigan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Giovanni LoGrasso Department of Ophthalmology, Visual and Anatomical Sciences, Wayne State University School of Medicine/Kresge Eye Institute , Detroit, Michigan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Linda D. Hazlett Department of Ophthalmology, Visual and Anatomical Sciences, Wayne State University School of Medicine/Kresge Eye Institute , Detroit, Michigan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Shunbin Xu Department of Ophthalmology, Visual and Anatomical Sciences, Wayne State University School of Medicine/Kresge Eye Institute , Detroit, Michigan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Shunbin Xu For correspondence: sxu{at}med.wayne.edu Abstract Full Text Info/History Metrics Preview PDF Abstract Purpose The miR-183/96/182 cluster (miR-183C) is required for normal functions of sensory neurons (SN) and various immune cells, including myeloid cells (MC). This research aims to reveal the roles of miR-183C of SN in the interplay of corneal sensory nerves (CSN) and MC during Pseudomonas aeruginosa (PA) keratitis. Methods Double-tracing mice with SN-specific (SNS) conditional knockout of miR-183C (CKO) and age-and sex-matched wild type (WT) controls were used. Their CSN are labeled with Red Fluorescent Protein (RFP); MC with Enhanced Green (EG)FP. The left corneas were scarified and infected with ATCC19660 PA. Corneal flatmounts were prepared at 3, 6, and 12 hours post-infection (hpi) and 1, 3, and 5 days (d)pi for confocal microscopy. Myeloperoxidase (MPO) assay and plate count were performed at 1 dpi. Results In WT mice, CSN began to degenerate as early as 3 hpi, starting from the fine terminal CSN in the epithelial/subepithelial layers, most prominently in the central region. By 1 dpi, CSN in the epithelium/subepithelial layer were nearly completely destroyed, while stromal nerves persisted. From 3 dpi, CSN were obliterated in both layers. In CKO vs WT mice, CNS followed a slightly slower pace of degeneration. CSN density was decreased at 3 and 6 hpi. However, at 3 dpi, residual large-diameter stromal CSN were better preserved. MC were decreased in the central cornea at 3 and 6 hpi, but increased in the periphery. Both changes were more prominent in CKO vs WT mice. At 12 hpi, densely packed MC formed a ring-shaped band circling a “dark” zone nearly devoid of MC, colocalizing with CSN most degenerated zone in the central cornea. In CKO vs WT, the ring center was larger with fewer MC. At 1 dpi, the entire cornea was filled with MC; however, MC density was lower in CKO mice. An MPO assay showed decreased neutrophils in PA-infected cornea of CKO mice. This led to a decreased severity of PA keratitis at 3 dpi. Conclusions This double-tracing model reveals the interplay between CSN and MC during PA keratitis with greater clarity, providing new insights into PA keratitis. CSN-imposed modulation on innate immunity is most impressive within 24 hours after infection. Functionally, the miR-183C in CSN modulates CSN density and the dynamics of MC fluxes-a neuroimmune interaction in display. Introduction Pseudomonas aeruginosa (PA) is a Gram-negative bacterium and a common causative organism associated with contact lens-related disease in developed countries 1 . PA-induced keratitis is one of the most rapidly developing and destructive diseases of the cornea 1 . It is estimated that 140 million people wear contact lenses worldwide, making PA keratitis a global cause of visual impairment and blindness 1 – 5 . Currently, PA keratitis is mainly treated by topical administration of antibiotics; in severe cases, subconjunctival injection may be employed. Although antibiotic treatment reduces the bacterial burden, tissue damage often occurs as a result of a poorly controlled host immune response 6 , 7 . Additionally, frequent emergence of antibiotic resistant strains poses serious challenges for the effective management of the disease 8 – 10 . Deeper insights into the pathogenesis will be the keys to the development of alternative treatment of the disease. The cornea is the most densely sensory innerved tissue in the body 11 – 13 . The neuronal cell bodies of corneal sensory nerves (CSN) reside in the trigeminal ganglion (TG) and can be activated by PA 14 . Evidences suggest that CSN secrete neuropeptides, e.g., substance (s) P and Calcitonin Gene-Related Peptide (CGRP), which modulate the immune response to PA infection 14 – 19 . Although a few studies indicated that CSN were quickly degenerated upon PA infection 14 , 20 , the data in these reports were limited and fragmented and lacked details of the dynamic changes of CSN. The simultaneous changes of the CSN and innate immune cells have not been studied hindering deeper understanding of the mechanisms of neuroimmune interaction during PA keratitis. microRNAs (miRNAs) are small, endogenous, non-coding RNAs and are important post-transcriptional regulators of gene expression 21 – 24 . They play an important role in human diseases 25 – 32 and are viable therapeutic targets 33 – 36 . However, their role in bacterial keratitis remain largely unexplored. Previously, we identified a conserved, paralogous miRNA cluster, the miR-183/96/182 cluster (referred to as miR-183C from here on), which is highly expressed in all primary sensory neurons of all major sensory domains, including the trigeminal ganglia (TG), where sensory neurons innervating the cornea reside 37 – 39 . Inactivation of the miR-183C disrupts the highly organized whorl-like pattern of the subbasal plexus of the CSN and results in decreased CSN density, sensitivity to mechanical stimuli and reduced levels of neuropeptides in the cornea 39 , 40 . Furthermore, we discovered that the miR-183C is also expressed in innate myeloid cells (MC), including corneal resident myeloid cells (CRMC) 41 as well as circulating MC, e.g., macrophage (Mφ) and polymorphonuclear leukocytes (PMN) 39 . miR-183C regulates the production of pro-inflammatory cytokines in response to PA infection by these cells and their phagocytosis and bacterial killing capacity through targeting key genes involved in related pathways 39 – 42 . Complete inactivation of miR-183C in a conventional knockout mouse model results in decreased immune/inflammatory response and reduced severity of PA keratitis 39 . Topical application of anti-miR-183C in the cornea leads to a reversible CSN regression, enhanced functional maturation of infiltrating MC and a decreased severity of PA keratitis 43 , suggesting that miR-183C is a therapeutic target for the treatment of PA keratitis. Recently, we developed a sensory neuron-specific (SNS) and a myeloid cell-specific (MS) conditional miR-183C knockout mouse models 40 . Using these models, we demonstrated that, in naïve mice, miR-183C imposes both intrinsic and extrinsic regulations on CSN, CRMC and corneal function through cell type-specific target genes 40 . These data support the hypothesis that miR-183C modulates corneal responses to PA infection through its dual regulation of CSN and innate immune cells and neuroimmune interaction. In the SNS-CKO mouse model, CSN are clearly labeled with Nav1.8-Cre-induced SNS expression of TdTomato red fluorescent protein (RFP), while the MC with Csf1r-promoter-driven enhanced green fluorescent protein (EGFP) 40 . This double-tracing mouse model provides an unprecedented powerful tool to simultaneously monitor CSN and MC, including both resident and infiltrating MC 40 . Here we report our observations of the simultaneous, dynamic changes of CSN and MC during PA keratitis and the impact of SNS-CKO of miR-183C. Our data provide new insights into the neuroimmune interaction and the pathogenesis of PA keratitis and the roles of miR-183C in CSN in the development of the disease. Methods Mice All experiments and procedures involving mice and their care were pre-reviewed and approved by the Wayne State University Institutional Animal Care and Use Committee and carried out in accordance with National Institute of Health and Association for Research in Vision and Ophthalmology (ARVO) guidelines. The study is reported in accordance with ARRIVE guidelines. Euthanasia was performed by cervical dislocation under anesthesia with isoflurane followed by thoracotomy. Mice with miR-183C CKO allele, the miR-183C f/f , were provided by Dr. Patrick Ernfors, Karolinska Institutet, Sweden through the European Mouse Mutant Archive (EMMA. ID: EM12387). The miR183C f allele has two loxP sites flanking the 5’ and 3’ ends of the miR-183C for robust Cre-mediated miR-183C knockout 44 . The sensory nerve-specific Nav1.8-Cre mice 45 were kindly provided by Dr. John N. Wood, University College London, through Dr. Theodore J. Price, University of Texas, Dallas. This strain is now available at the Jackson Laboratory (Stock Number: 036564). The voltage-gated sodium channel Na v 1.8 (encoded by the Scn10a gene) is one of the signature genes of the majority of nociceptive sensory neurons in the TG and dorsal root ganglia (DRG) 14 , 46 – 48 . Na v 1.8 promoter-driven Cre recombinase (Na v 1.8-Cre) is expressed in nearly all corneal nociceptive sensory nerves 14 , 45 , 49 . The Csf1r-EGFP or MacGreen mice 50 were purchased from the Jackson lab (Stock number. 018549) In this strain, the EGFP transgene is under the control of the 7.2-kb mouse colony stimulating factor 1 receptor (Csf1r ) promoter, allowing specific expression of EGFP in the mononuclear phagocyte system (MPS) myeloid cells, including monocytes, Mj and dendritic cells (DCs) 50 , 51 . The reporter strain, R26 LSL-RFP(+/+) mice 52 , also known as Ai14 (Stock number: 007914, the Jackson Laboratory) has a loxP-flanked STOP cassette (LSL) in front of a tdTomato RFP cassette, all of which are inserted into the ROSA26 locus 52 . The LSL prevents the transcription of tdTomato RFP; however, when Cre recombinase is present, the LSL cassette will be excised to allow the expression of tdTomato RFP. SNS-CKO mice [Na v 1.8-Cre(+/-);miR-183C f/f ;R26 LSL-RFP(+/+) ;Csf1r-EGFP(+/+)] and their WT littermates [Na v 1.8-Cre(+/-);miR-183C +/+ ;R26 LSL-RFP(+/+) ;Csf1r-EGFP(+/+)] were produced by breeding of Na v 1.8-Cre (+/-), miR-183C f/f , R26 LSL-RFP and Csf1r-EGF(+/-) mice. All breeding showed a normal mendelian inheritance pattern. 8-20 weeks old, male or female mice were used separately in all experiments. The age, sex and number of the mice in each experiment are specified in the figure legends and/or the text. On average, 5 mice/sex/genotype was used at each time-point for various analyses. PA infection PA infection was conducted as we described previously 39 , 41 , 43 . Briefly, mice were anesthetized with isoflurane in a well-ventilated hood. The cornea of the left eye was scarified with a 25 5/8-gauge needle. Three 1-mm incisions were made to the corneal surface, which penetrated the epithelial layer, but no deeper than the superficial stroma. 5.0 × 10 6 colony forming units (CFU) PA (strain 19660; ATCC) in a 5-μl volume was topically delivered. Corneal disease was graded at 1, 3 and 5 days-post-infection (dpi) using an established scale 15 : 0, clear or slight opacity, partially or fully covering the pupil; +1, slight opacity, covering the anterior segment; +2, dense opacity, partially or fully covering the pupil; +3, dense opacity, covering the entire anterior segment; and +4, corneal perforation. Photography with a slit lamp was used to illustrate disease at 1, 3 and 5 dpi. The corneas were harvested at 3, 6, and 12 hours post-infection (hpi) and 1, 3, and 5 days post-infection (dpi) for corneal flatmount preparation or other assays as detailed below. Corneal flatmount preparation Corneal flatmount was performed as described previously 40 , 41 , 43 . Briefly, mice were euthanized; eyes were enucleated and transferred to cold 1% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB), pH 7.4 for 10 minutes before being carefully dissected under a dissection microscope (VWR International, Radnor, PA). The cornea was carefully dissected out and fixed in cold 1% PFA in 0.1 M PB, pH 7.4 for another hour (h) on ice. The cornea was flattened by six evenly spaced cuts from the periphery toward the center and mounted in Vectashield media with DAPI (Vector Laboratories, Burlingame, CA) on Superfrost Plus slides (Fisherbrand). Quantification of corneal nerve density and myeloid cells All slides were imaged using a TCS SP8 laser confocal microscope (Leica Microsystems Inc., Buffalo Grove, IL). To capture the images of the entire cornea, a series of Z-stacked images under 10x objective were taken across the entire cornea and stitched together. All images were taken and processed under the same parameters, e.g., laser power and digital gain and offset, without saturation to ensure that all images were comparable and the differences among samples reflected their biological differences. These stitched Z-stacked images were merged/flattened for cell counting or CSN density quantification using Adobe Photoshop CS6 (64 bit) and ImageJ 1.52p software ( http://imagej.nih.gov/ij . NIH, Bethesda, MD, USA) as described previously 41 . Briefly, the raw image was first converted to the 16-bit grayscale; then the black & white binary image is optimized for the threshold to faithfully represent the original image and the cell density. The EGFP+ cells in the entire cornea were counted for corneas at 3 and 6 dpi. For corneas at 12 hpi and 1, 3, and 5 dpi, since the number of EGFP+ MC are too densely packed to be distinguished individually, we cannot reliably count the numbers; instead, we measured the volumes of fluorescence of comparable areas (Adobe Photoshop CS6) as relative quantification of MC for comparisons between WT and CKO samples. To quantify CSN density, the mean value of pixels in a 500x500 μm 2 square covering the whorl center of the subbasal plexus was quantified as the nerve density of the center. In the periphery, three 500x500 μm 2 squares were randomly placed and measured; and the average was recorded as CSN density of the peripheral cornea. Myeloperoxidase (MPO) assay A myeloperoxidase (MPO) assay was performed as described before to quantify the number of neutrophils 39 , 43 . Briefly, corneas were harvested, homogenized in 1 ml potassium phosphate buffer (50 mM, pH 6.0) containing 0.5% hexadecyltrimethyl-ammonium bromide (Sigma-Aldrich). After four freeze/thaw cycles, 100 μl supernatant was added to 2.9 ml o-dianisidine dihydrochloride substrate buffer (16.7 mg/ml) with 0.0005% hydrogen peroxide. The change in absorbance at 460 nm was read every 30 seconds (s) for 5 min on a Helios-α spectrophotometer (ThermoFisher). Units of MPO per cornea were calculated: 1 unit of MPO activity is equivalent to ∼2 x 10 5 PMN 53 . Quantitation of viable bacteria Bacteria were quantitated by plate count assay as described before 39 , 43 . Briefly, each cornea was homogenized in 1.0 ml sterile saline containing 0.25% BSA. 0.1 ml of the corneal homogenate was serially diluted 1:10 in the same solution and selected dilutions were plated in triplicate on Pseudomonas isolation agar (Becton Dickinson). Plates were incubated overnight (O/N) at 37 °C and the number of colonies counted. Results are reported as log 10 number of CFU/cornea ± SEM. Statistical analyses were performed as we described previously 40 . Briefly, When the comparison was made among more than 2 conditions, one-way ANOVA with Bonferroni’s multiple comparison test was employed (GraphPad Prism); adjusted p<0.05 was considered significant. Otherwise, a two-tailed Student’s t test was used to determine the significance; p<0.05 was considered significant. Each experiment was repeated at least once to ensure reproducibility and data from a representative experiment are shown. Quantitative data is expressed as the mean ± SEM. Results Dynamics of corneal sensory nerve degeneration during PA keratitis To uncover the behaviors of CSN during PA infection, we infected (ATCC 19660) the left eyes of SNS-CKO and age-and sex-matched WT control mice and performed flatmount confocal imaging at 3, 6, and 12 hpi and 1, 3, and 5 dpi. Our data showed that CSN began to degenerate as early as 3 hpi, the earliest time-point of our study, in both WT and SNS-CKO mice ( Fig.1 . *: p<0.05 in Fig.1E ). At 3 hpi, significant reduction of CSN density occurred first in the central region of the infected vs non-infected contralateral cornea; however, no significant change was observed in the peripheral cornea in both WT and SNS-CKO mice ( Fig.1A -E ). Download figure Open in new tab Figure 1. CSN began to degenerate as early as 3 hpi. A-D . Representative compressed flatmount confocal images of the corneas of PA infected (B,D) and non-infected contralateral eyes (A,C) of WT (A,B) and SNS-CKO mice (12-20 weeks old, female). OD: oculus dexter (right eye); OS: oculus sinister (left eye). Scale bars: 500 μm. E . Quantification of sensory nerve density of the central and peripheral regions of the cornea of WT and SNS-CKO mice. CL: contralateral eye; I: infected eye. *: p<0.05. In WT mice, as the disease developed, the degeneration of CSN gradually worsened from the central region to the periphery and from the fine, terminal nerves in the epithelial/subepithelial layer to the larger stromal nerves ( Fig.2A , blue line Fig.3 ). In the peripheral region, CSN degenerated in a slower pace when compared to the central region; significant degeneration did not occur until 12 hpi [ Fig.2A-c , Fig.3B (^: p<0.05, 12 hpi vs 3 hpi)]. From 12 hpi to 1 dpi, CSN continued to degenerate in both central and peripheral regions [ Fig.2A , Fig.3 (‡: p<0.05 1 dpi vs 12 hpi)]. By 1 dpi, most of the fine terminal nerves in the epithelial and subepithelial layer were degenerated, while most of the stromal nerves were still preserved ( Fig.2A -d ). From 1 to 3 dpi, CSN degeneration in the peripheral region drastically accelerated in WT (blue line in Fig.3B . ∏: p<0.05, 3 dpi vs 1 dpi). From 3 dpi, all sensory nerves were nearly completely destroyed in both the central and peripheral regions of the WT mice ( Fig 2.Ae,f ; Fig.3 ). CSN in the contralateral eyes did not have significant changes at all times (data not shown). Download figure Open in new tab Figure 2. Representative compressed flatmount confocal images of the corneas of PA infected left eyes of WT ( A ) and SNS-CKO mice ( B ) at 3, 6 and 12 hpi and 1, 3 and 5 dpi. Scale bars: 500 μm. Download figure Open in new tab Figure 3. Dynamic changes of CSN density of the central ( A ) and peripheral regions of the corneas ( B ) of SNS-CKO and their age-and sex-matched WT controls at 3, 6, and 12 hours (h) and 1, 3, and 5 days post-infection (d), in comparison to the ones of the non-infected, contralateral eye at 3 hpi [3 h (CL)]. *: p<0.05, WT vs CKO; #: p<0.05, vs 3 h CL; ^: p<0.05, vs 3 hpi; †: p<0.05, vs 6 hpi; ‡: p<0.05, vs 12 hpi. ∏: p<0.05, vs 1 dpi. n= 5, 4, 6, 5, 5, and 5 for WT, and 3, 4, 5, 6, 5, and 5 for SNS-CKO at 3, 6, and 12 hpi, 1, 3, and 5 dpi, respectively. Inactivation of miR-183C in sensory neurons resulted in more severe reduction of CSN in the epithelial/subepithelial layer in the early stages but enhanced preservation of the large CSN in the stromal layer at later stages Previously, we have shown that inactivation of miR-183C in CSN but not in myeloid cells results in decreased CSN density in naïve CKO vs WT control mice 40 . Consistent with this observation, at the early stage of PA infection (3 hpi), CSN density was significantly decreased in the SNS-CKO vs WT controls, in both the central and peripheral regions ( Fig.1 -3 ). However, CSN in CKO mice appeared to degenerate at a slower pace in the central region of the cornea in the early stage of the disease, when compared to the WT mice ( Fig.3A ). By 6 hpi, the sensory nerve density in the central region showed no significant difference between the SNS-CKO and WT control mice, because of faster degeneration in the WT mice ( Fig.3A ). Thereafter, CSN degeneration in the central region followed a similar course in both SNS-CKO and WT mice at 12 hpi, 1, 3 and 5 dpi ( Fig.2 ; Fig.3A ). In the peripheral region of SNS-CKO mice ( Figs.2B; orange line in Fig.3B ), CSN degeneration followed a slower pace than the central region (orange line in Fig.3A ). The CSN density remained relative stable in the early stages of PA infection (3 and 6 hpi)( Fig.3B ). Similar as in the naïve mice that we reported earlier 40 , the CSN density was significantly decreased in the SNS-CKO vs WT control mice ( Figs.1, 2, 3B. *: p<0.05). CSN density in the peripheral region did not show a significant decrease until 12 hpi in WT mice (blue line in Fig.3B ); however, it remained stable at this time-point in the SNS-CKO mice (orange line in Fig.3B ), erasing the difference of CSN density between SNS-CKO and WT control mice ( Fig.3B ). From 12 hpi to 1 dpi, SNS-CKO and WT mice showed similar pace of CSN degeneration in the peripheral regions of the cornea ( Fig.3B ). However, from 1 to 3 dpi, CSN density did not further decrease in the SNS-CKO mice, while in the WT mice it continued to degenerate and were nearly completely destroyed by 3 dpi ( Fig.2A-e ,B-e; Fig.3B ). This resulted in higher sensory nerve density in the SNS-CKO vs WT controls at 3 dpi ( Fig.2A -e,B-e; Fig.3B . *: p<0.05). By 5 dpi, CSN were completely degenerated in the both SNS-CKO and WT controls ( Fig.2A -f,B-f; Fig.3B ). Inactivation of miR-183C in the CSN resulted in changes of the dynamics of myeloid cells in the cornea during PA keratitis To study the dynamics of myeloid cells during PA keratitis, we quantified the number of Csf1r-EGFP+ myeloid cells in the cornea at 3, 6, 12 hpi and 1, 3, and 5 dpi. At early stages of PA keratitis (3 and 6 hpi), in the WT mice, although the total number of Csf1r-EGFP+ myeloid cells per cornea showed little difference between the infected vs non-infected contralateral control eyes ( Fig.4A ,B ), the density of myeloid cells in the central region of the cornea was significantly decreased, however, slightly increased in the peripheral region ( Fig.4A ,C,D . *: p<0.05). The decreased myeloid cell density in the central region is reminiscent of the macrophage/leukocyte disappearance reaction (M/LDR) that we reported early 40 . Download figure Open in new tab Figure 4. Comparison of myeloid cell number and distribution in the corneas of the SNS-CKO vs WT control mice at 3 hpi. A . Representatives of compressed flatmount confocal images of the infected left eyes (OS) (b,d) and non-infected contralateral eyes (OD) (a,c) of the SNS-CKO (c,d) and WT control mice (a,b). B . Quantification of the total number of Csf1r-EGFP+ myeloid cells (MC number)/cornea in the infected left eyes (OS, inf) and non-infected contralateral right eyes (OD, CL) of the SNS-CKO and WT control mice. C,D . MC number per 500 x 500 μm 2 square in the central (C) and peripheral regions of the cornea (D). Unlike the WT mice, in the SNS-CKO mice, the total number of myeloid cells in the cornea was increased in the infected vs non-infected contralateral eyes as early as 3 hpi, suggesting accelerated activation of immune defense machinery and faster infiltration of myeloid cells from the circulation. Densely packed Csf1r-EGFP+ myeloid cells formed infection foci ( Fig.4A-d ), which were not seen in the WT control mice at this stage ( Fig.4A-b ), suggesting accelerated infiltration in the SNS-CKO mice. In spite of the enhanced infiltration of myeloid cells in the peripheral regions of the cornea, like in the WT mice, the density of myeloid cells in the central region of the infected vs non-infected, contralateral control cornea was also decreased in the SNS-CKO mice ( Fig.4A,B ), suggesting the M/LDR was not affected by SNS-CKO of miR-183C, consistent with our previous report 40 . At 6 hpi, myeloid cells in the infected eyes of SNS-CKO mice remained at a similar level as 3 hpi, while the number of myeloid cells in the corneas of infected eyes of WT mice was further increased to a similar level as the SNS-CKO mice ( Fig.5A,B ). Similar to the SNS-CKO mice, densely packed infection foci appeared in the infected corneas of the WT mice ( Fig.5A-b ), although the sizes of foci appeared to be smaller than in the SNS-CKO mice ( Fig.5A-b &d ). Download figure Open in new tab Figure 5. Comparison of myeloid cell number and distribution in the corneas of the SNS-CKO vs WT control mice at 6 hpi. A . Representatives of compressed flatmount confocal images of the infected left eyes (OS) (b,d) and non-infected contralateral eyes (OD) (a,c) of the SNS-CKO (c,d) and WT control mice (a,b). Scale bars: 500 μm. B . Quantification of the total number of Csf1r-EGFP+ myeloid cells (MC number)/cornea. C,D . MC number per 500 x 500 μm 2 square in the central (C) and peripheral regions of the cornea (D). From 6 to 12 hpi, drastic change occurred in the infected corneas ( Fig.6 ). At 12 hpi, in both SNS-CKO and WT mice, densely-packed myeloid cells formed a ring-shaped band circling a “dark zone” in the center of the cornea, which was nearly devoid of myeloid cells, and appeared to coincide with the central area of the cornea where CSN had degenerated (yellow circles in Fig.6A ). Since it is impossible to count the number of EGFP+ myeloid cells, we quantified the volume of the EGFP fluorescence to reflect the density of myeloid cells between SNS-CKO and WT controls. Our data showed that at 12 hpi, although the overall of intensity of EGFP signals showed no difference between SNS-CKO, vs WT controls ( Fig.6B ), the size of the central “dark zone” was larger in the SNS-CKO vs WT mice, consistent with enhanced CSN degeneration ( Fig.6A,C ), while the density of myeloid cells in the central “dark zone” were decreased in SNS-CKO vs WT controls ( Fig.6A,D . *: p<0.05). Download figure Open in new tab Figure 6. Comparison of myeloid cell distribution in relation to CSN in the corneas of the SNS-CKO vs WT control mice at 12 hpi. A . Representatives of compressed flatmount confocal images of the myeloid cells (GFP+, a,d), sensory nerves (RFP+, b,e) and merged images (c,f) of the corneas of infected eyes of the SNS-CKO (d-f) and WT control mice (a-c). Scale bars: 500 μm. B . Quantification of the average intensity of EGFP fluorescence of the whole cornea. C,D. The area (C) and average intensity of the EGFP fluorescence of the MC (D) in the “dark zone” encircled by the inflammatory ring. Scale bars: 500 μm. At 1 dpi, the entire corneas appeared to be filled with densely packed myeloid cells in both SNS-CKO and WT control mice ( Fig.7 ). However, the average intensity of EGFP in the CKO mice was decreased ( Fig.7B ) and the area filled with EGFP+ myeloid cells was reduced ( Fig.7C ), when compared to the WT mice, suggesting decreased infiltration of myeloid cells in the cornea. Download figure Open in new tab Figure 7. Comparison of myeloid cell distribution in relation to CSN in the corneas of the SNS-CKO vs WT control mice at 1 dpi. A . Representatives of compressed flatmount confocal images of the myeloid cells (GFP+, a,d), CSN (RFP+, b,e) and merged images (c,f) of the corneas of infected eyes of the SNS-CKO (d-f) and WT control mice (a-c). Scale bars: 500 μm. B,C . Quantification of the average intensity (B) and the area of EGFP fluorescence of the whole cornea (C). D,E. The number of PMN (D) and residual bacteria per cornea at 1 dpi (E). To test this, we performed MPO assay to quantify the number of neutrophils in the infected cornea. Our result showed that the number of neutrophils in the SNS-CKO mice was indeed significantly decreased (∼1.4 fold) when compared to the WT mice at 1 dpi ( Fig.7D ). It is estimated that the SNS-CKO mice has ∼7 million less neutrophils/cornea than the WT, based that 1 unit of MPO is equivalent to approximately 2x10 5 neutrophils 53 . Consistently, a viable bacterial plate assay to quantify the residual bacteria in the infected cornea of SNS-CKO mice showed slightly increased number of colony forming units (CFU) of PA, when compare to the WT controls (∼2.3 fold increase and ∼ 4.7 x 10 5 more bacteria/cornea), although the data did not pass the threshold of statistical significance ( Fig.7E ). From 3 dpi ( Fig.8A-B ), the entire cornea was filled with EGFP+ myeloid cells. No quantitative difference between WT and CKO was observed. Download figure Open in new tab Figure 8. Myeloid cell distribution in relation to CSN in the corneas of the SNS-CKO vs WT control mice at 3 dpi (A) and 5 dpi (B). Inactivation of miR-183C in the sensory neurons resulted in a decrease of the severity of PA keratitis at 3 dpi In spite the decreased neutrophil infiltration in the SNS-CKO vs WT mice at 1 dpi, the disease severity did not show significant difference at this time-point ( Fig.9 ). However, at 3 dpi, PA keratitis showed a slight decreased severity in the SNS-CKO mice ( Fig.9 ). At this stage, the infected corneas of 8 out of 9 WT mice were perforated; while only 4 out of 10 SNS-CKO mice were perforated; the clinical scores of the rest 6 SNS-CKO remained at +3 ( Fig.9 ). At 5 dpi, no significant difference in clinical scores was observed between the SNS-CKO and WT control mice ( Fig.9 ). Download figure Open in new tab Figure 9. SNS-CKO of miR-183C resulted in slightly decreased severity of PA keratitis at 3 dpi. A . Representative slit lamp photographs of SNS-CKO and WT control mice at 1, 3 and 5 dpi. B . Clinical scores. n numbers at each time-point and genotype are specified at the bottom. Discussion and Conclusion The miR-183C is required for the normal functions of both CSN and innate immune cells 39 – 42 . Complete inactivation of miR-183C in mice has significant functional impacts on both corneal sensory innervation and innate immunity, resulting in decreased CSN density and a disrupted structural pattern of the subbasal plexus of the cornea 39 . It also reduces pro-inflammatory responses while enhancing the phagocytic and bacterial killing capacity of innate immune cells, including Mφ and neutrophils 39 , 42 . These lead to an overall decreased severity of PA keratitis in the miR-183C conventional KO vs WT control mice 39 . Consistently, corneal knockdown of miR-183C by anti-miR-183C application to the cornea showed similar effect 43 , suggesting miR-183C modulates PA keratitis through its dual regulation of CSN and innate immunity. Here, to further specify the contribution of its regulation of sensory innervation to the modulation of PA keratitis, we created a SNS-CKO model. In comparison to their WT controls, our data showed that inactivation of SNS-CKO of miR-183C resulted in changes in the dynamics of sensory nerve degeneration (intrinsic regulation) as well as innate immune cell infiltration in the cornea (extrinsic regulation) - a display of the effect of neuroimmune interactions during PA keratitis. The severity of the disease in the SNS-CKO vs WT control mice was only modestly decreased when compared to what we observed in the conventional KO mice 39 or acute miR-183C knockdown 43 , in which miR-183C is inactivated 39 or down-regulated in both sensory nerves and innate immune cells 43 . This observation suggests that effects of knockout/knockdown of miR-183C in sensory nerves and innate immunity are additive, if not synergistic; in combination, they result in more robust decrease of the severity of PA keratitis. Simultaneous knockdown of miR-183C in CSN and myeloid cells is required for a therapeutic approach. A myeloid cell-specific CKO of miR-183C mouse model will help to further elucidate whether and how miR-183C’s functions in sensory nerves and innate immune cells work additively or synergistically in modulation of PA keratitis. Although it has been reported that CSN are destroyed quickly after PA infection 14 , 20 , previous studies were conducted by immunostaining of pan-neuronal marker, β-III tubulin, and did not provide clear images of the entire cornea with spatial and temporal details 14 , 20 . Therefore, the behavior of the sensory innervation during PA keratitis remain unresolved. In the current study, the double tracing mouse model, in which corneal sensory nerves are labeled with Nav1.8-Cre driven RFP reporter, while myeloid cells are labeled with Csf1r-EGFP, provides a powerful tool to simultaneously observe the behaviors and sensory nerves and innate immune cells and their interactions during PA keratitis with unprecedented resolution. Our study provides new insights into the dynamic changes of CSN during PA keratitis with spatial and temporal details. We showed that CSN degeneration began as early as 3 hpi, starting with fine terminal nerves in the epithelial/subepithelial layers in the central region of the cornea. The CSN degeneration gradually progressed to the periphery of the cornea. By 1 dpi, sensory nerves in the epithelial layer were nearly completely destroyed, however, large-diameter nerves and their branches in the stromal layer persisted. Since CSN modulate corneal immune/inflammatory responses through exocytosis at their nerve endings, which are mostly distributed in the epithelial and subepithelial plexus 12 , 54 , 55 , based on our result, it is reasonable to speculate that modulation of the immune response to PA infection by the sensory nerves occurs mostly in the first 24-hour window. However, we cannot exclude the possibility that sensory nerves remodel their synaptic distribution during PA keratitis and modulate the disease process in later stages. Previously, we and other have shown that miR-183C is responsible for the terminal functional differentiation and organization in sensory neurons of various sensory organs 38 , 56 – 59 . Consistently, complete inactivation or knockdown as well as SNS-CKO of miR-183C result in decreased sensory serve density and interrupted patterns of the fine terminal nerves in the epithelial and subbasal plexus of the cornea of naïve KO or SNS-CKO mice when compared to their corresponding WT controls 39 , 40 , 43 . This suggests that miR-183C promotes corneal sensory neurite growth and patterning during corneal development and the establishment of the CSN innervation. This effect is a result of its direct regulation of the molecules involved in axon guidance and neuronal projection-related genes 40 , 43 . Our observation of decreased CSN density in the SNS-CKO mice in the early stage of the PA keratitis is possibly a reflection of this effect. However, our data also suggest that the CSN showed an accelerated degeneration in the WT mice in the early stage of PA keratitis; by 6 hpi, the difference in the sensory nerve density between the SNS-CKO and WT controls were “erased”. At 3 dpi, the residual large sensory nerves were better preserved in the SNS-CKO vs WT control mice. These results support a hypothesis that, in the context of PA keratitis, downregulation of miR-183C may have a protective effect on the large-diameter nerves in the stroma. Consistent with this hypothesis, our previously published transcriptomic data suggests that, in addition to neuron projection development-related genes, miR-183C regulates neuronal survival/apoptosis through targeting a series of key molecules in these processes 40 . Future studies will be needed to directly test this hypothesis. Besides the behavior of CSN, our study also provides new insights into the dynamics of innate immune cells during PA keratitis. Here we showed that at the early stages of the disease (3 and 6 hpi), the density of myeloid cells was decreased in the central region of the cornea, while increased in the peripheral region. The decrease in the myeloid cell density in the central region is possibly a result of the M/LDR that we reported earlier 41 ; while the increased density in the peripheral region of the cornea a result of infiltration. M/LDR is considered to allow the immune-regulatory resident Mφ to temporarily “give way” to pro-inflammatory myeloid cells to evoke an acute inflammatory response before they “re-appear” to contain the inflammation and promote tissue repair 60 – 62 . Our data supports this hypothesis. However, the current mouse model cannot distinguish between the resident myeloid cells from the infiltrating cells. Future studies with specific lineage tracing capability will further elucidate this. Furthermore, our data, for the first time, simultaneously revealed the dynamic changes of CSN and myeloid cells during PA keratitis, providing an opportunity to uncover the interactions of CSN and innate immune cells. Consistent with our previous report 41 , our data showed that in the early stage of PA keratitis (3 and 6 hpi), infiltration of myeloid cells was accelerated in the SNS-CKO vs WT control mice, suggesting an extrinsic regulation of miR-183C in CSN of the innate immune responses. It is possibly a result of the direct regulations of miR-183C on pro-inflammatory neuropeptides, including Cx3cl1, which is known to promote chemotactic migration of microglial in the central nervous system 63 , 64 , and mediate the homing of resident myeloid cells in the cornea 65 and recruitment of Mφ in other tissues 66 . Additional studies will be needed to further test this hypothesis. At 12 dpi, the densely-packed infiltrating myeloid cells formed a ring-shaped band, which we designate here as an “inflammatory ring”. This inflammatory ring circled a zone in the central region of the cornea, which was nearly devoid of myeloid cells, coinciding with the central region of the cornea where CSN degenerated first. The number of myeloid cells was decreased in the ring center of the SNS-CKO vs WT controls, while CSN density was reduced, suggesting a contribution of CSN degeneration in the formation of the “inflammatory ring” – neuroimmune interaction. The inflammatory ring appeared to be a transient phenomenon, as, by 1 dpi, the infected cornea including the central region was filled with myeloid cells. The functional significance of the inflammatory ring during PA keratitis warrants further studies. It is well known that different mouse strains with different genetic background have their differences in corneal nerve densities 67 and immune responses to PA infection 68 – 71 . In the current study, all mice were on the C57BL/6 background, which is known to favor Th1 responsiveness and has enhanced susceptibility and severe PA keratitis 69 . Mice with different genetic backgrounds are known to have different immune/inflammatory responses in PA keratitis. For example, mouse strains with Th2-dominant response, e.g., BALB/c, are relatively resistant to PA infection and have a milder course of disease without corneal perforation 68 – 71 . Additional studies are required to determine whether our observations of the neuroimmune responses here apply to other mouse models and as well as in humans. A few limitations of the current study need to be addressed in our future endeavors. First, although the flatmount microscopic study illustrated greater details of the simultaneous changes of CSN and myeloid cells during PA keratitis, they are static images of neuro-immune components at different stages of the disease. Time-lapse live imaging by advanced multiphoton microscopy will be required to provide further insights of neuroimmune interactions. Second, in this study, we focused on the interaction of sensory neurons and innate immune cells. However, increasing evidence suggests corneal sympathetic nerves interact with CSN, CRICs and other cellular components and modulate corneal homeostasis and pathogenesis of various corneal diseases. For example, the sympathetic nerves have been shown to promote inflammatory responses and delay corneal re-epithelialization after epithelial debridement 72 – 74 . Inhibition of sympathetic innervation alleviated acute tobacco smoke induced deficiencies in corneal wound healing 75 . In herpes stromal keratitis (HSK), it has been shown that CD4+ T cell-and myeloid cell-produced VEGF causes sensory nerve regression and simultaneously enhances sympathetic innervation in the cornea 76 . The sympathetic neurotransmitter, norepinephrine (NE), has been shown to aggravate bacterial keratitis by a combination of compromising epithelial integrity, enhancing bacterial growth and virulence, and inflammatory responses 77 – 79 . In addition, our recent systems biology approach using single-cell RNA sequencing technology showed that miR-183C’s regulation is not restricted to the immune cells and sensory nerves, but also imposes global modulation of the entire corneal cellular landscape 80 . Studies of the simultaneous changes and interactions among CSN, sympathetic nerves, innate immunes and other components of the cornea with a systems biology approach are warranted. 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