an increased risk of als is associated with certain populations who have a history of extensive physical contact such as varsity athletics , professional soccer players , and military veterans [ 13 ] .
motor nerve injury as a trigger to degeneration has been proposed in these populations , but the underlying mechanism remains elusive . in order to investigate this hypothesis , we utilized the motor nerve injury ( facial nerve axotomy ( fna ) ) in the als mouse model ( sod1 mice ) to evaluate the impact of fna on motoneuron survival after injury .
we found that fna - induced motor neuron loss is significantly increased in sod1 mice relative to wt mice .
importantly , the increased motor neuron loss in sod1 mice can be prevented by adoptive transfer of immune cells from wild - type mice .
these data suggest that individuals with a genetic susceptibility to als are more vulnerable to nerve injury - induced neurodegeneration . because such vulnerability is impacted by the immune system , we hypothesize that fna may induce a more pronounced proinflammatory response in sod1 mice than in wt mice , which in turn impairs the function of neuroprotective immune responses . as the pivotal cell of immunoregulation , the cd4 t cell has been of a great interest in the investigation of the pathogenesis of als .
cd4 t cells have several subsets with distinct immunoregulatory functions . in late - stage als
patients , the total number of nave cd4 t cells is decreased and cd4 t cell infiltration in the spinal cord and brain is significantly increased [ 5 , 6 ] .
in addition , elevated th1 cells in cerebrospinal fluid and elevated il-17 and th17-related cytokines ( il-6 , tnf- , il-1 , and il-23 ) [ 79 ] in the serum have also been observed in als patients .
furthermore , the level of antineuroinflammatory subsets , th2 and treg cells [ 10 , 11 ] , appear to regulate the speed of disease progression
. however , the roles of proneuroinflammatory subsets , th1 and th17 cells , in promoting als development has yet to be established due to two challenges : late diagnosis and the chronic nature of the disease .
the late diagnosis makes it difficult to conclude whether observed abnormal immune response and inflammation are the cause or the result of the disease .
the chronic nature of als also makes it difficult to determine the best timing for the detection of such autoimmune responses .
we have previously demonstrated that fna is capable of inducing a readily detectable immune response in a predictable time period ( 714 days ) . in the current study , we performed fna in presymptomatic b6sjl sod1 mice ( 8-week - old ) and examined cd4 t responses in a time course after fna .
we found that abnormal cd4 t cell activation with increased th17 cells is present in sod1 mice prior to the onset of neurological symptoms .
these results suggest that sod1 mice have impaired immunoregulatory mechanisms that normally dampen injury - induced inflammatory responses and that th17 cell - promoted inflammation might contribute to the increase in injury - induced motoneuron death in sod1 mice .
six - week - old female b6sjl sod1 and wild - type female b6sjlf1/j mice were obtained from jackson laboratory ( sacramento , ca , usa ) .
all mice were housed and surgery was performed as previously described [ 4 , 12 ] .
all surgical procedures were completed in accordance with national institutes of health guidelines on the care and use of laboratory animals for research purposes .
the right cervical lymph nodes ( draining cervical lymph node ( dcln ) ) were collected from uninjured ( control ) or axotomized mice ( n = 4/group ) at 7 , 9 , and 14 days postaxotomy ( dpa ) .
cd4 t cells were isolated via automacs using anti - cd4 magnetic beads as previously described [ 4 , 12 ] .
cd4 t cells separated from the draining cervical lymph node preparation were incubated for 6 hours with phorbol myristate acetate ( pma , 50  ng / ml ) and ionomycin ( 500  ng / ml , p / i , sigma , st .
louis , mo ) with brefeldin a ( bfa , 10  g / ml , sigma , st .
louis , mo ) added during the final 2 hours . for the intracellular staining , cd4 t cells were permeabilized with saponin ( 1  mg / ml , sigma , st .
louis , mo ) and double - stained with two of the following antigens : anti - cd4-efluor 450 , anti - ifn--alexa fluor-488 , anti - il-17a - pe - cy7 , anti - tnf--pe , anti - il-10-percp - cy5.5 and anti - il-4-apc , or anti - cd8-fitc . for the immune cells
subsets identification , freshly collected dcln cells were stained with anti - cd3-pe , anti - b220-apc - cy7 , anti - cd4-fitc , and anti - cd8-apc .
for identification of activation , cells were stained with anti - cd4-efluor 450 , anti - cd69-percp - cy5.5 , anti - cd44-apc , and anti - cd62l - fitc .
a one - way anova with the bonferroni post hoc test was used for comparisons of two specific groups .
head injury is associated with an increased risk for developing als [ 13 ] , leading us to hypothesize that inappropriate activation of the immune system from prior injury may underlie the development of als .
therefore , in the current study , we used the fna model of motor neuron injury to compare immune responses in wt versus sod1 mice which serve as a mouse model of als to examine underlying alterations in immune activation and implications for disease development in sod1 mice ( presymptomatic , 8-week - old b6sjl ) .
as shown in figure 1(a ) , basal numbers ( prior to the fna ) of total cells recovered from one dcln wt mouse were 6.13  0.44 ( 10 ) versus 12.1  0.99 ( 10 ) for sod1 mice , suggesting that sod1 mice have greater baseline number of lymphocytes than do wt mice .
following fna , a transient increase in the number of total cells recovered was noted in wt mice and returned to basal levels at 14 days after fna .
in contrast , sod1 mice showed a progressive and sustained increase in total cell numbers in the dcln .
differences in cell counts correlated with the size of dcln in these mice ( figure 1(b ) ) . to further differentiate t cell subsets
, we analyzed the percentage of cd4 versus cd8 t cells ( figures 1(c)1(e ) ) .
prior to the fna , both wt and sod1 mice had a ratio of cd4  :  cd8 that was approximately 2  :  1 .
although the ratio of cd4  :  cd8 remained close to 2  :  1 in wt mice following fna , it decreased to a ratio approaching 1  :  1 in sod1 mice ( figures 1(c)-1(d ) ) .
however , because total cell numbers in the dcln increased after fna , the absolute number of both cd4 and cd8 increased in both wt and sod1 ( figure 1(e ) ) .
these data suggest that an enhanced basal level of inflammation in sod1 mice may impair immunoregulatory mechanisms that normally dampen injury - induced inflammatory responses before the onset of neurological symptoms .
accordingly , we examined further the activation status of cd4 t cell responses at 7 days after fna , the peak time of cd4 t cell response . in the wt mice ,
t cells at both the early activation stage ( cd69 , figures 1(f)1(h ) ) and effector stage ( cd62lcd44 , figures 1(i)1(k ) ) were increased after fna , as reflected by frequency ( figures 1(g ) and 1(j ) ) and total number ( figures 1(h ) and 1(k ) ) .
the same pattern was also found in sod1 mice , but at a higher magnitude .
in addition , fna - induced activation levels of cd4 t cells in wt mice were comparable to that of sod1 mice prior to the fna in terms of both percentage and total number , suggesting that the fna - induced activation of t cells in wt mice may occur in a similar manner as t cell activation in sod1 mice , prior to disease onset .
cd4 t subsets have distinct functions in terms of neuroprotective or neurodestructive effects [ 1015 ] .
therefore , we examined dcln cd4 t subsets in wt and sod1 mice at 7 days after fna by staining intracellularly for ifn- ( th1 cells ) , il-17 ( th17 cells ) , tnf- ( pro - inflammatory cells ) , il-4 ( th2 cells ) , and il-10 ( treg cells ) . following fna , the frequency of th1 cells in the wt mice was decreased , although the total number did not significantly change . in contrast , both the frequency and total number of th1 cells were significantly increased in sod1 mice ( figures 2(a)-2(b ) , first column ) .
in addition , th17 cells in the wt mice did not change in frequency and increased only slightly in total number .
in contrast , sod1 mice possessed both a greater frequency and more total th17 cells than did wt mice , regardless of fna state .
in fact , the frequency of th17 cells was 3-fold higher ( 0.65  0.05% versus 0.13  0.03% ) prior to injury and was 4-fold higher ( 0.77  0.02% versus 0.16  0.02% ) after fna ( figure 2(b ) , middle column ) in sod1 mice , whereas the numbers of total th17 cells were 9-fold higher [ 2.8  0.31 versus 0.28  0.03 ( 10 ) ] prior to injury and 5-fold higher [ 5.39  0.61 versus 0.89  0.14 ( 10 ) ] after fna ( figure 2(c ) middle column ) in sod1 mice .
in the wt mice , the frequency and total number of th1th17 cells were similar prior to and after fna .
in contrast , the frequency and total number of th1th17 cells in sod1 mice decreased after fna .
we did not find significant difference of th2 and treg cells between wt and sod1 mice prior to or after fna ( data not shown ) .
further analysis of tnf- expression in cd4 t cells revealed that both frequency and percentage of tnf--single - positive cells were significantly increased in the wt mice following fna .
however , in sod1 mice , though the percentage did not change after fna , the total number of tnf- cells significantly increased ( figures 2(e ) and 2(f ) , left column ) .
tnf--expressing th17 ( tnf-th17 ) in wt mice was low in both frequency and total number and did not significantly change after fna .
in contrast , tnf-th17 in sod1 mice had a higher basal frequency and total number which was further increased in response to fna ( figures 2(e ) and 2(f ) , right column ) .
these data suggest that tnf--expressing th17 cells might be an important subset of autoimmune cells involved in injury - induced inflammatory damage in sod1 mice .
we have previously demonstrated that cd4 t cells mediate neuroprotection after nerve injury [ 4 , 16 ] . in als
, cd4 t cells also play an important role in restricting disease progression [ 13 , 14 ] ; however , this neuroprotection is in a context- and subset - dependent manner [ 1015 ] .
for example , anti - inflammatory subsets of cd4 t cells are generally thought to be the types of neuroprotective immune cells which support facial motoneuron survival after nerve injury and may therefore slow down the disease progression in als [ 10 , 11 ] .
our previous studies revealed that multiple subsets of cd4 t cells develop following fna in wt mice , including both anti - inflammatory and proinflammatory subsets of cd4 t cells .
we hypothesize that the balance between these subsets of cd4 t is critical for the resolution of necessary and beneficial inflammation as well as induction of repair tissue and support mechanisms for survival of damaged motoneurons . in the current work
, we used the sod1 mice , an als mouse model , to show that fna induces significant motoneuron loss relative to wt mice and that this loss is similar to that of immunodeficient mice ( rag2 mice ) [ 4 , 16 ] .
exacerbation of fna - induced motoneuron loss in sod1 mice may result from a poorly controlled inflammatory response to injury , increased basal levels of inflammation inherent to this model or perhaps to failure of development of neuroprotective cd4 t subsets   as the current study revealed that there are significant differences of fna - induced immune responses between wt and sod1 mice .
first , we found that while wt mice mounted a well - controlled immune response in the draining lymph nodes following fna , sod1 mice had an enhanced and persistent immune response as indicated by the enlarged size of the draining lymph nodes as well as an increase in total cell numbers .
further analysis for t cell subset responses revealed that the ratio of cd4  :  cd8 t cells in wt mice did not change in response to fna , whereas the ratio decreased in sod1 mice . because cd4 t cells regulate cd8 t cell responses and cd4 t cells are the major type of t cells that impact the neuronal survival after fna , we focused on cd4 t cell responses in the analysis of t cell activation and subsets .
our data indicate that activation of cd4 t cells is higher in sod1 than wt mice . regarding the subsets
, we did not find significant differences in anti - inflammatory t cell subsets ( th2 and treg cells ) between wt and sod1 mice either prior to or after fna ; however , significant differences were noted in the proinflammatory subsets ( th1 and th17 cells ) between these two types of mice . without nerve injury ,
th1 cell frequency and total number were similar in wt and sod1 mice ; however , fna induced an increase in th1 total number in sod1 mice but not in wt mice . in the uninjured state ,
both th17 cell frequency and total number were higher in sod1 mice than in wt mice .
further increases in th17 cell frequency and total number were noted in sod1 mice but not in wt mice under fna injury conditions .
importantly , th17 cells are also tnf--expressing cells in sod1 mice but not in wt mice .
these data collectively indicate that feed - forward proinflammatory response to injury occurs in sod1 mice .
we recently demonstrated that whole splenocytes , but not isolated cd4 t cells , from wt mice can reduce fna - induced facial motor nucleus ( fmn ) loss in sod1 mice .
in addition , isolated cd4 t cells , but not whole splenocytes collected from sod1 mice , are capable of supporting fmn survival in immunodeficient mice ( rag2 ) .
these data suggest that the microenvironment in sod1 mice may direct cd4 t cell differentiation into a neurodestructive subset .
consistent with this hypothesis , the data from the current study suggest that fna - induced th17 cell responses in sod1 mice may exacerbate neuroinflammation , without a concomitant induction of the neural repair phase , which in turn results in increased motoneuron death .
enhanced cd4 t cell activation and th17 cell responses in sod1 mice exist prior to the onset of overt neurological symptoms . motor nerve injury further increases cd4 t cell activation and th17 cell responses in sod1 mice .
we therefore propose that cell - promoted inflammation may be involved in the motoneuron death during als disease onset and progression .
 an increased risk of als has been reported for veterans , varsity athletes , and professional football players . 
 the mechanism underlying the increased risk in these populations has not been identified ; however , it has been proposed that motor nerve injury may trigger immune responses which , in turn , can accelerate the progression of als . accumulating evidence indicates that abnormal immune reactions and inflammation are involved in the pathogenesis of als , but the specific immune cells involved have not been clearly defined . to understand how nerve injury and immune responses may contribute to als development , we investigated responses of cd4 + t cell after facial motor nerve axotomy ( fna ) at a presymptomatic stage in a transgenic mouse model of als ( b6sjl sod1g93a ) . 
 sod1g93a mice , compared with wt mice , displayed an increase in the basal activation state of cd4 + t cells and higher frequency of th17 cells , which were further enhanced by fna . in conclusion , 
 sod1g93a mice exhibit abnormal cd4 + t cell activation with increased levels of th17 cells prior to the onset of neurological symptoms . 
 motor nerve injury exacerbates th17 cell responses and may contribute to the development of als , especially in those who carry genetic susceptibility to this disease . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusion