Respiratory Syncytial Virus G Protein CX3C Motif Impairs Human Airway Epithelial and Immune Cell Responses

Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory infection in infants and young children and causes disease in the elderly and persons with compromised cardiac, pulmonary, or immune systems. Despite the high morbidity rates of RSV infection, no highly effective treatment or vaccine is yet available. The RSV G protein is an important contributor to the disease process. A conserved CX3C chemokine-like motif in G likely contributes to the pathogenesis of disease. Through this motif, G protein binds to CX3CR1 present on various immune cells and affects immune responses to RSV, as has been shown in the mouse model of RSV infection. However, very little is known of the role of RSV CX3C-CX3CR1 interactions in human disease. In this study, we use an in vitro model of human RSV infection comprised of human peripheral blood mononuclear cells (PBMCs) separated by a permeable membrane from human airway epithelial cells (A549) infected with RSV with either an intact CX3C motif (CX3C) or a mutated motif (CX4C). We show that the CX4C virus induces higher levels of type I/III interferon (IFN) in A549 cells, increased IFN-α and tumor necrosis factor alpha (TNF-α) production by human plasmacytoid dendritic cells (pDCs) and monocytes, and increased IFN-γ production in effector/memory T cell subpopulations. Treatment of CX3C virus-infected cells with the F(ab′)₂ form of an anti-G monoclonal antibody (MAb) that blocks binding to CX3CR1 gave results similar to those with the CX4C virus. Our data suggest that the RSV G protein CX3C motif impairs innate and adaptive human immune responses and may be important to vaccine and antiviral drug development.     

Roflumilast Inhibits Respiratory Syncytial Virus Infection in Human Differentiated Bronchial Epithelial Cells

Respiratory syncytial virus (RSV) causes acute exacerbations in COPD and asthma. RSV infects bronchial epithelial cells (HBE) that trigger RSV associated lung pathology. This study explores whether the phosphodiesterase 4 (PDE4) inhibitor Roflumilast N-oxide (RNO), alters RSV infection of well-differentiated HBE (WD-HBE) in vitro. WD-HBE were RSV infected in the presence or absence of RNO (0.1-100 nM). Viral infection (staining of F and G proteins, nucleoprotein RNA level), mRNA of ICAM-1, ciliated cell markers (digital high speed videomicroscopy, β-tubulin immunofluorescence, Foxj1 and Dnai2 mRNA), Goblet cells (PAS), mRNA of MUC5AC and CLCA1, mRNA and protein level of IL-13, IL-6, IL-8, TNFα, formation of H₂O₂ and the anti-oxidative armamentarium (mRNA of Nrf2, HO-1, GPx; total antioxidant capacity (TAC) were measured at day 10 or 15 post infection. RNO inhibited RSV infection of WD-HBE, prevented the loss of ciliated cells and markers, reduced the increase of MUC5AC and CLCA1 and inhibited the increase of IL-13, IL-6, IL-8, TNFα and ICAM-1. Additionally RNO reversed the reduction of Nrf2, HO-1 and GPx mRNA levels and consequently restored the TAC and reduced the H₂O₂ formation. RNO inhibits RSV infection of WD-HBE cultures and mitigates the cytopathological changes associated to this virus.     

CX3CR1 Is Expressed by Human B Lymphocytes and Meditates CX3CL1 Driven Chemotaxis of Tonsil Centrocytes

Background

Fractalkine/CX₃CL1, a surface chemokine, binds to CX₃CR1 expressed by different lymphocyte subsets. Since CX₃CL1 has been detected in the germinal centres of secondary lymphoid tissue, in this study we have investigated CX₃CR1 expression and function in human naïve, germinal centre and memory B cells isolated from tonsil or peripheral blood.

Methodology/Principal Findings

We demonstrate unambiguously that highly purified human B cells from tonsil and peripheral blood expressed CX₃CR1 at mRNA and protein levels as assessed by quantitative PCR, flow cytometry and competition binding assays. In particular, naïve, germinal centre and memory B cells expressed CX₃CR1 but only germinal centre B cells were attracted by soluble CX₃CL1 in a transwell assay. CX₃CL1 signalling in germinal centre B cells involved PI3K, Erk1/2, p38, and Src phosphorylation, as assessed by Western blot experiments. CX₃CR1⁺ germinal centre B cells were devoid of centroblasts and enriched for centrocytes that migrated to soluble CX₃CL1. ELISA assay showed that soluble CX₃CL1 was secreted constitutively by follicular dendritic cells and T follicular helper cells, two cell populations homing in the germinal centre light zone as centrocytes. At variance with that observed in humans, soluble CX₃CL1 did not attract spleen B cells from wild type mice. OVA immunized CX₃CR1⁻/⁻ or CX₃CL1⁻/⁻ mice showed significantly decreased specific IgG production compared to wild type mice.

Conclusion/Significance

We propose a model whereby human follicular dendritic cells and T follicular helper cells release in the light zone of germinal centre soluble CX₃CL1 that attracts centrocytes. The functional implications of these results warrant further investigation.     

Respiratory Syncytial Virus Can Infect Basal Cells and Alter Human Airway Epithelial Differentiation

Respiratory syncytial virus (RSV) is a major cause of morbidity and mortality worldwide, causing severe respiratory illness in infants and immune compromised patients. The ciliated cells of the human airway epithelium have been considered to be the exclusive target of RSV, although recent data have suggested that basal cells, the progenitors for the conducting airway epithelium, may also become infected in vivo. Using either mechanical or chemical injury models, we have demonstrated a robust RSV infection of p63⁺ basal cells in air-liquid interface (ALI) cultures of human bronchial epithelial cells. In addition, proliferating basal cells in 2D culture were also susceptible to RSV infection. We therefore tested the hypothesis that RSV infection of this progenitor cell would influence the differentiation status of the airway epithelium. RSV infection of basal cells on the day of seeding (MOI≤0.0001), resulted in the formation of an epithelium that showed a profound loss of ciliated cells and gain of secretory cells as assessed by acetylated α-tubulin and MUC5AC/MUC5B immunostaining, respectively. The mechanism driving the switch in epithelial phenotype is in part driven by the induced type I and type III interferon response that we demonstrate is triggered early following RSV infection. Neutralization of this response attenuates the RSV-induced loss of ciliated cells. Together, these data show that through infection of proliferating airway basal cells, RSV has the potential to influence the cellular composition of the airway epithelium. The resulting phenotype might be expected to contribute towards both the severity of acute infection, as well as to the longer-term consequences of viral exacerbations in patients with pre-existing respiratory diseases.     

Vaccination To Induce Antibodies Blocking the CX3C-CX3CR1 Interaction of Respiratory Syncytial Virus G Protein Reduces Pulmonary Inflammation and Virus Replication in Mice

Respiratory syncytial virus (RSV) infection causes substantial morbidity and some deaths in the young and elderly worldwide. There is no safe and effective vaccine available, although it is possible to reduce the hospitalization rate for high-risk children by anti-RSV antibody prophylaxis. RSV has been shown to modify the immune response to infection, a feature linked in part to RSV G protein CX3C chemokine mimicry. This study determined if vaccination with G protein polypeptides or peptides spanning the central conserved region of the G protein could induce antibodies that blocked G protein CX3C-CX3CR1 interaction and disease pathogenesis mediated by RSV infection. The results show that mice vaccinated with G protein peptides or polypeptides containing the CX3C motif generate antibodies that inhibit G protein CX3C-CX3CR1 binding and chemotaxis, reduce lung virus titers, and prevent body weight loss and pulmonary inflammation. The results suggest that RSV vaccines that induce antibodies that block G protein CX3C-CX3CR1 interaction may offer a new, safe, and efficacious RSV vaccine strategy.Human respiratory syncytial virus (RSV) is an important and ubiquitous respiratory virus causing serious lower respiratory tract diseases in infants and young children and substantial morbidity and mortality in the elderly and immunocompromised (7, 11, 20, 21). Despite substantial efforts to develop safe and effective RSV vaccines, none have been successful. The first RSV candidate vaccine, a formalin-inactivated alum-precipitated RSV (FI-RSV) preparation, did not confer protection and was associated with a greater risk of serious disease with subsequent natural infection (9, 60). Live attenuated and inactivated whole virus vaccine candidates have also failed to protect, as they were either insufficiently attenuated or demonstrated the potential for enhanced pulmonary disease upon subsequent RSV infection (6, 37, 39, 41, 45). Similarly, subunit vaccine candidates, such as purified F protein and a prokaryotically expressed fusion protein comprising a fragment of the RSV G protein (residues 130 to 230) fused by its N terminus to the albumin binding domain of streptococcal protein G (designated BBG2Na), have been shown to be inadequate (8, 33, 37, 41). The specific reasons for RSV vaccine failure remain to be answered but could be related to RSV-mediated circumvention of immunity and, more broadly, to the lack of durable immunity elicited in response to natural RSV infection, as people of all ages may experience repeated infections and disease throughout life (3, 41, 45).Evidence indicates that the RSV F protein is important in inducing protective immunity (19, 38), but studies evaluating a BBG2Na vaccine candidate in combination with different adjuvants and by different routes of administration have shown a role for G protein in protection against RSV in rodents (4, 10, 17, 32, 43, 44, 49, 51). The structural elements of the G protein fragment in the BBG2Na vaccine candidate implicated in protective efficacy were mapped, and five different B-cell epitopes were determined, i.e., residues 145 to 159, 164 to 176, 171 to 187, 172 to 187, and 190 to 204 (44, 48). Interestingly, immunogenicity of peptides with residues 145 to 159 was dependent on the orientation of the covalent peptide coupling to the carrier proteins, as mice vaccinated with C-terminally coupled peptides developed protective antibody titers, whereas mice vaccinated with N-terminal peptides did not. The focus of the BBG2Na vaccine studies centered on development of protective neutralizing antibodies, and the studies showed that vaccination or priming with the G protein fragment in BBG2Na did not induce signs of enhanced pulmonary pathology (17, 42, 46, 50).Despite the strong evidence that G protein peptides and polypeptides can induce protective immunity, the G protein has also been implicated in disease pathogenesis (30, 40, 41, 54). One of the disease mechanisms linked to the G protein is CX3C chemokine mimicry (56). RSV G protein has marked similarities to fractalkine, the only known CX3C chemokine, including similarities in structural features (56). Both G protein and fractalkine exist as membrane-bound and secreted forms, and both contain a CX3C chemokine motif that can bind to the fractalkine receptor, CX3CR1 (15, 27). Fractalkine functions to recruit immune cells to sites of inflammation, in particular, CX3CR1⁺ leukocytes, which include subsets of NK cells and CD4⁺ and CD8⁺ T cells (23). RSV G protein has been shown to have fractalkine-like leukocyte chemotactic activity in vitro (56). In vivo, RSV G protein acts as a fractalkine antagonist, modulating the immune response to infection by inhibiting fractalkine-mediated responses by altering the trafficking of CX3CR1⁺ cells and modifying the magnitude and cadence of cytokine and chemokine expression (23, 55). Infection of mice with a mutant RSV lacking the CX3C motif leads to a substantial increase of pulmonary NK cells and CD4⁺ and CD8⁺ cells compared to infection with wild-type RSV (23). This suggests that G protein CX3C-CX3CR1 interaction contributes to immune evasion and may contribute to disease pathogenesis. Thus, G protein CX3C interaction with CX3CR1 is an important target for disease intervention strategies against RSV infection.In the present study, we investigated a new RSV vaccine strategy, using G protein polypeptide and peptide vaccination to generate antibodies reactive to the central conserved cysteine noose region of the G protein to block G protein CX3C motif interaction with CX3CR1. We hypothesize that vaccines inducing G protein-CX3CR1 blocking antibodies will prevent much of the RSV G protein-mediated immune modulation and disease pathogenesis. Our results show that antibodies induced by the central conserved noose region of the G protein block G protein binding to CX3CR1, prevent body weight loss indicative of disease pathogenesis, decrease pulmonary inflammation, and decrease lung virus titers compared to antibodies reactive to N- and C-terminal regions of the G protein. These results suggest that a vaccine strategy to induce G protein CX3C-CX3CR1 blocking antibodies may be useful to prevent G protein-mediated immune modulation and disease pathogenesis.     

Respiratory syncytial virus G protein and G protein CX3C motif adversely affect CX3CR1+ T cell responses

Interactions between fractalkine (CX3CL1) and its receptor, CX3CR1, mediate leukocyte adhesion, activation, and trafficking. The respiratory syncytial virus (RSV) G protein has a CX3C chemokine motif that can bind CX3CR1 and modify CXCL1-mediated responses. In this study, we show that expression of the RSV G protein or the G protein CX3C motif during infection is associated with reduced CX3CR1+ T cell trafficking to the lung, reduced frequencies of RSV-specific, MHC class I-restricted IFN-gamma-expressing cells, and lower numbers of IL-4- and CX3CL1-expressing cells. In addition, we show that CX3CR1+ cells constitute a major component of the cytotoxic response to RSV infection. These results suggest that G protein and the G protein CX3C motif reduce the antiviral T cell response to RSV infection.     

Fractalkine Is Expressed in Early and Advanced Atherosclerotic Lesions and Supports Monocyte Recruitment via CX3CR1

Fractalkine (CX3CL1, FKN) is expressed in the inflamed vascular wall and absence of FKN reduces atherogenesis. Whether FKN is expressed throughout all stages of atherosclerotic disease and whether it directly contributes to monocyte recruitment to atherosclerotic lesions is not known. We collected human atherosclerotic plaque material and blood samples from patients with carotid artery disease undergoing endarterectomy. Plaques were analyzed by immunohistochemistry and qPCR. We found that FKN is expressed at all stages of atherosclerotic lesion formation, and that the number of FKN-expressing cells positively correlates with the number of CX3CR1-positive cells in human carotid artery plaques. In the circulation, soluble FKN levels are significantly elevated in the presence of high-grade (sub-occlusive) stenosis. To determine the role of the FKN-CX3CR1 axis for monocyte adhesion in vivo we then performed intravital videofluorescence microscopy of the carotid artery in ApoE(-/-) mice. Notably, FKN-CX3CR1 interactions are critical for recruitment of circulating monocytes to the injured atherosclerotic vascular wall. Thus, this chemokine dyad could represent an attractive target for anti-atherosclerotic strategies.     

人呼吸道合胞病毒感染的A549细胞中长链非编码RNA表达谱研究

目的:探讨人呼吸道合胞体病毒(human respiratory syncytial virus,RSV)感染A549细胞的长链非编码RNA(long non-coding RNAs,lncRNA)表达谱的差异,更好地了解宿主与RSV之间的相互作用的可能分子机制.方法:1 MOI RSV感染A549细胞,24h后提取R...     

CX3CR1 Signaling on Monocytes Is Dispensable after Intracerebral Hemorrhage

Intracerebral hemorrhage is a subset of stroke for which there is no specific treatment. The Ly6C^hi CCR2⁺ monocytes have been shown to contribute to acute injury after intracerebral hemorrhage. The other murine monocyte subset expresses CX3CR1 and lower Ly6C levels, and contributes to repair in other disease models. We hypothesized that the Ly6C^lo CX3CR1⁺ monocytes would contribute to recovery after intracerebral hemorrhage. Intracerebral hemorrhage was modeled by blood injection in WT and CX3CR1-null bone marrow chimeras. Neurological outcomes and leukocyte recruitment were quantified at various time points. Functional outcomes were equal at 1, 3, 7, and 14 days after intracerebral hemorrhage in both genotypes. No differences were observed in leukocyte recruitment between genotypes on either 3 or 7 days after intracerebral hemorrhage. A few hundred Ly6C^lo monocytes were found in the ipsilateral hemisphere in each genotype and they did not change over time. Peripherally derived CX3CR1⁺ monocytes were observed in the perihematomal brain 7 and 14 days after intracerebral hemorrhage. Our data suggests CX3CR1 signaling on monocytes does not play an influential role in acute injury or functional recovery after intracerebral hemorrhage and therefore CX3CR1 is not a therapeutic target to improve outcome after intracerebral hemorrhage.     

Identification of a Linear Heparin Binding Domain for Human Respiratory Syncytial Virus Attachment Glycoprotein G

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract disease in infants and young children worldwide. Infection is mediated, in part, by an initial interaction between attachment protein (G) and a highly sulfated heparin-like glycosaminoglycan (Gag) located on the cell surface. Synthetic overlapping peptides derived from consensus sequences of the G protein ectodomain from both RSV subgroups A and B were tested by heparin-agarose affinity chromatography for their abilities to bind heparin. This evaluation identified a single linear heparin binding domain (HBD) for RSV subgroup A (184A-->T198) and B (183K-->K197). The binding of these peptides to Vero cells was inhibited by heparin. Peptide binding to two CHO cell mutants (pgsD-677 and pgsA-745) deficient in heparan sulfate or total Gag synthesis was decreased 50% versus the parental cell line, CHO-K1, and decreased an average of 87% in the presence of heparin. The RSV-G HBD peptides were also able to inhibit homologous and heterologous virus infectivity of Vero cells. These results indicate that the sequence 184A/183K-->198T/K197 for RSV subgroups A and B, respectively, defines an important determinant of RSV-G interactions with heparin.     

Acute and Chronic Airway Disease After Human Respiratory Syncytial Virus Infection in Cotton Rats (Sigmodon hispidus)

Infection with respiratory syncytial virus (RSV) generally presents as a mild, upper airway disease in human patients but may cause severe lower airway disease in the very young and very old. Progress toward understanding the mechanisms of RSV pathogenesis has been hampered by a lack of relevant rodent models. Mice, the species most commonly used in RSV research, are resistant to upper respiratory infection and do not recapitulate the pattern of virus spread in the human host. To address the need for better rodent models of RSV infection, we have characterized the acute and chronic pathology of RSV infection of a relatively permissive host, cotton rats (Sigmodon hispidus). We demonstrate that virus delivered to the upper airway results in widespread RSV replication in the ciliated respiratory epithelial cells of the nasal cavity and, to a lesser extent, of the lung. Although acute inflammation is relatively mild and rapidly eliminated after viral clearance, chronic, eosinophilic lung pathology persists. These data support the use of cotton rats as a robust rodent model of human RSV disease, including the association between RSV pneumonia and subsequent development of allergic asthma.Abbreviations: BAL, bronchoalveolar lavage; RSV, human respiratory syncytial virusHuman respiratory syncytial virus (RSV) is the primary cause of lower airway disease in infants and children worldwide.³⁹ Although generally limited to the upper airway, RSV infection can also manifest as bronchiolitis and pneumonia in young children and the elderly and has been implicated as a major cause of middle ear infections (otitis media).^3,55 In addition to the problems associated with acute illness, children who experience severe RSV disease in infancy are at increased risk for development of asthma and recurrent wheezing later in childhood.^35,53,56RSV is ubiquitous worldwide, with most children infected in infancy, and essentially all children are infected by 3 y of age.²⁰ Although reinfection throughout life has been documented, the true incidence of upper airway infection is difficult to quantify, given that treatment for such infections typically is not sought. In comparison, lower airway infection is more likely to come to medical attention, and it is currently estimated that at least 33.8 million episodes of RSV-induced acute pulmonary infection in children younger than 5 y occur yearly and that as many as 200,000 of those episodes are fatal.⁴⁰ Although supportive treatment for severe RSV disease is highly effective in infants, access to such treatment is generally only available in industrialized countries. More than 90% of fatal RSV cases occur in the developing world.⁴⁰Mice have been used extensively to study RSV infection, yet there are key limitations of the mouse model for the study of human RSV disease and immunity. The most important of these is the poor permissiveness of the mouse for human RSV. RSV replicates to a limited extent in the mouse lung, and large viral loads delivered in a relatively large volume are generally used with this model. Even with intranasal inoculation, primarily lower, and not upper, airway infection is achieved in WT mice. This scenario is unlike the human disease, in which the upper airway is the primary target of RSV replication.^24,25 Mice are essentially resistant to upper airway infection, with little to no virus detected in nasal washes evaluated by plaque assay, and only rare RSV-antigen–positive cells are detected by immunohistochemistry.^13,18,21 In addition to this altered route of entry, the pattern of lung infection is markedly divergent between humans and mice. In humans, RSV primarily infects ciliated bronchiolar epithelial cells and, to a much lesser extent, alveolar cells.³² In contrast, bronchiolar epithelial cells are infected only rarely in the mouse lung; instead RSV targets the pneumocytes.³⁷Unlike mice, cotton rats are susceptible to RSV infection of the upper airway, and the pattern of lower airway infection mirrors that seen in human patients. The cotton rat was established as a model of RSV infection more than 3 decades ago and has since emerged as the preferred rodent model in which to evaluate RSV therapeutics and vaccine candidates.^{19,23,33,41,49,57,60} In addition, as the availability of species-specific reagents has improved, cotton rats have become an increasingly useful model in which to study RSV pathogenesis.⁶ However, whereas the susceptibility of cotton rats to RSV infection has been established firmly, the pathology of RSV infection in this species has not yet been extensively characterized.Asthma clearly is a multifactorial disease, dependent on both genetic and environmental factors (see references 28 and 30 for recent reviews), and many studies have pointed to a role for respiratory virus infection in the induction of asthma. In one study,¹⁷ the combination of atopy and the presence of virus in nasal secretions synergistically increased the odds ratio for wheezing in children 25-fold, and another study²⁹ showed that repeated rhinovirus infections in the first 3 y of life increased by 26-fold the risk of developing asthma by the age of 6 y. This relationship is obviously a complex one, influenced both by the nature and the timing of the viral infection. Nonetheless, as many as 80% of acute asthma exacerbations in children and approximately 50% in adults are associated with viral infection.^33,42 The majority of these infections are attributed to rhinoviruses, but other respiratory viruses, including influenza virus, RSV, and coronaviruses, can provoke these attacks.^33,42 Beyond the exacerbation of established asthma, evidence that severe RSV disease in infancy is correlated with development of asthma and recurrent wheezing in later childhood is mounting. An association between lower airway RSV infection and subsequent development of recurrent wheezing and asthma was demonstrated more than 30 y ago,¹⁶ and several recent prospective studies have strengthened this correlation.^27,53In the current study, we expand upon existing knowledge of the cotton rat model of RSV infection by characterizing the spread of virus after droplet inhalation and the early and late inflammatory response to viral challenge. We confirm the observation that cotton rats are relatively permissive in regard to RSV infection as compared with mice, and we demonstrate that the infection of cotton rats, like that in the human host, is primarily an upper airway phenomenon. In addition, we show that primary RSV infection of the cotton rat lung, even at the low levels in this study, induces chronic changes consistent with those in cases of human allergic asthma. This finding is of interest because of the substantial literature associating early, severe respiratory infection with the development of asthma later in life. The studies of RSV infection in cotton rats that we describe here suggest that this species may be predisposed to atopy and that, long after virus clearance, changes associated with allergic inflammation persist in human hosts. Therefore, in addition to the usefulness of cotton rats for testing RSV treatments and vaccine candidates, we suggest that this species may serve as a model system for determining the involvement of virus infection in the development of allergic asthma.     

TMPRSS2 Isoform 1 Activates Respiratory Viruses and Is Expressed in Viral Target Cells

The cellular protease TMPRSS2 cleaves and activates the influenza virus hemagglutinin (HA) and TMPRSS2 expression is essential for viral spread and pathogenesis in mice. Moreover, severe acute respiratory syndrome coronavirus (SARS-CoV) and other respiratory viruses are activated by TMPRSS2. However, previous studies on viral activation by TMPRSS2 focused on a 492 amino acids comprising form of the protein (isoform 2) while other TMPRSS2 isoforms, generated upon alternative splicing of the tmprss2 mRNA, have not been characterized. Here, we show that the mRNA encoding a TMPRSS2 isoform with an extended N-terminal cytoplasmic domain (isoform 1) is expressed in lung-derived cell lines and tissues. Moreover, we demonstrate that TMPRSS2 isoform 1 colocalizes with HA and cleaves and activates HA. Finally, we show that isoform 1 activates the SARS-CoV spike protein for cathepsin L-independent entry into target cells. Our results indicate that TMPRSS2 isoform 1 is expressed in viral target cells and might contribute to viral activation in the host.     

CX3CR1 Is a Modifying Gene of Survival and Progression in Amyotrophic Lateral Sclerosis

The objective of this study was to investigate the association of functional variants of the human CX3CR1 gene (Fractalkine receptor) with the risk of Amyotrophic Lateral Sclerosis (ALS), the survival and the progression rate of the disease symptoms in a Spanish ALS cohort. 187 ALS patients (142 sporadic [sALS] and 45 familial) and 378 controls were recruited. We investigated CX3CR1 V249I (rs3732379) and T280M (rs3732378) genotypes and their haplotypes as predictors of survival, the progression rate of the symptoms (as measured by ALSFRS-R and FVC decline) and the risk of suffering ALS disease. The results indicated that sALS patients with CX3CR1 249^I/I or 249^V/I genotypes presented a shorter survival time (42.27±4.90) than patients with 249^V/V genotype (67.65±7.42; diff −25.49 months 95%CI [−42.79,−8.18]; p = 0.004; adj-p = 0.018). The survival time was shorter in sALS patients with spinal topography and CX3CR1 249^I alleles (diff = −29.78 months; 95%CI [−49.42,−10.14]; p = 0.003). The same effects were also observed in the spinal sALS patients with 249^I–280^M haplotype (diff = −27.02 months; 95%CI [−49.57, −4.48]; p = 0.019). In the sALS group, the CX3CR1 249^I variant was associated with a faster progression of the disease symptoms (OR = 2.58; 95IC% [1.32, 5.07]; p = 0.006; adj-p = 0.027). There was no evidence for association of these two CX3CR1 variants with ALS disease risk. The association evidenced herein is clinically relevant and indicates that CX3CR1 could be a disease-modifying gene in sALS. The progression rate of the disease''s symptoms and the survival time is affected in patients with one or two copies of the CX3CR1 249I allele. The CX3CR1 is the most potent ALS survival genetic factor reported to date. These results reinforce the role of the immune system in ALS pathogenesis.