Re: 염증기 3일 탐구!! 혈소판 유래 케모카인 --＞ 호중구, 근육재생 촉진 2023네이처!!

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Platelet-derived chemokines promote skeletal muscle regeneration by guiding neutrophil recruitment to injured muscles

• Article
• Open access
• Published: 22 May 2023

Platelet-derived chemokines promote skeletal muscle regeneration by guiding neutrophil recruitment to injured muscles

• Flavia A. Graca, 
• Anna Stephan, 
• Benjamin A. Minden-Birkenmaier, 
• Abbas Shirinifard, 
• Yong-Dong Wang, 
• Fabio Demontis & 
• Myriam Labelle 

Nature Communications volume 14, Article number: 2900 (2023) Cite this article

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Abstract

Skeletal muscle regeneration involves coordinated interactions between different cell types. Injection of platelet-rich plasma is circumstantially considered an aid to muscle repair but whether platelets promote regeneration beyond their role in hemostasis remains unexplored. Here, we find that signaling via platelet-released chemokines is an early event necessary for muscle repair in mice. Platelet depletion reduces the levels of the platelet-secreted neutrophil chemoattractants CXCL5 and CXCL7/PPBP. Consequently, early-phase neutrophil infiltration to injured muscles is impaired whereas later inflammation is exacerbated.

Consistent with this model, neutrophil infiltration to injured muscles is compromised in male mice with Cxcl7-knockout platelets. Moreover, neo-angiogenesis and the re-establishment of myofiber size and muscle strength occurs optimally in control mice post-injury but not in Cxcl7ko mice and in neutrophil-depleted mice. Altogether, these findings indicate that platelet-secreted CXCL7 promotes regeneration by recruiting neutrophils to injured muscles, and that this signaling axis could be utilized therapeutically to boost muscle regeneration.

초록

골격근 재생은 

다양한 세포 유형 간의 조화된 상호작용을 수반한다. 

혈소판 풍부 혈장 주사는 

근육 회복을 돕는 보조 수단으로 간주되지만,

 혈소판이 지혈 기능 이상의 재생 촉진 효과를 가지는지는 아직 밝혀지지 않았다. 

본 연구에서는 

혈소판이 분비하는 케모카인을 통한 신호전달이 

생쥐 근육 회복에 필수적인 초기 사건임을 확인하였다. 

혈소판 제거는 

혈소판이 분비하는 호중구 유인물질인 CXCL5 및 CXCL7/PPBP의 수준을 감소시킨다. 

Platelet depletion

reduces the levels of the platelet-secreted neutrophil chemoattractants CXCL5 and CXCL7/PPBP.

CXCL은 염증 부위로 백혈구를 유인하는 케모카인 계열의 단백질.
이 단백질은 염증 반응에서 중요한 역할을 하는 호중구와 T 림프구를 주로 유인.

호중구는 세균이나 곰팡이 감염에 대한 1차 방어선 역할을 하며,
T 림프구는 바이러스 감염이나 암세포 제거에 중요한 역할.

CXCL은
이러한 백혈구들을 염증 부위로 유인하여 면역 반응을 조절하고,
염증을 해결하는 데 도움

그 결과, 

손상된 근육으로의 초기 단계 호중구 침윤이 저해되는 

반면 후기 염증은 악화된다. 

이 모델과 일치하게, 

Cxcl7 결손 혈소판을 가진 수컷 생쥐에서는 

손상된 근육으로의 호중구 침윤이 손상된다. 

또한, 

신생혈관 형성 및 근섬유 크기 및 근력의 회복은 

대조군 생쥐에서는 손상 후 최적의 수준으로 이루어지지만, 

Cxcl7knockout 생쥐와 호중구 고갈 생쥐에서는 그렇지 않습니다. 

종합하면, 이러한 결과들은 

혈소판 분비 CXCL7이 손상된 근육으로 호중구를 모집함으로써 재생을 촉진하며, 

이 신호 전달 축이 근육 재생을 촉진하기 위한 치료적 목적으로 활용될 수 있음을 시사합니다.

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Introduction

Skeletal muscle has the remarkable capacity to repair injuries that occur in response to trauma, xenobiotics, and strenuous exercise1,2,3,4,5. In addition to physiological injuries, muscle damage and regeneration occurs in muscular dystrophies4,6,7 and cancer-induced cachexia8, and it is defective in aging9,10.

Previous studies have found that muscle regeneration starts immediately after the death of myofibers, which leads to the recruitment of immune cells to the muscle. These infiltrating cells are necessary for muscle regeneration via their capacity to remove cellular debris and promote myogenesis, i.e., the fusion of satellite muscle stem cells to form new myofibers2,3,11,12,13. Several waves of immune cell recruitment occur during muscle regeneration, and these include neutrophils, monocytes, macrophages, and T cells14. Neutrophils are recruited in the early phase of muscle regeneration and are followed by M1/M2 macrophages in later phases2,3,11,12,13. Whereas neutrophils and M1 macrophages contribute to inflammation, M2 macrophages have anti-inflammatory functions2,3,11,12,13,15. Partial depletion of neutrophils and macrophages impairs muscle regeneration16,17,18,19, indicating that invading immune cells are indeed necessary for muscle repair. Importantly, optimal muscle regeneration requires robust but transient recruitment of distinct immune populations, and the transition from an inflammatory to an anti-inflammatory state2,3,11,12,13. Consequently, persistent inflammation is a cause of myopathies and age-related muscle repair deficits20,21,22,23,24.

서론

골격근은

외상, 이물질, 격렬한 운동에 반응하여 발생하는 손상을 복구하는

놀라운 능력을 지니고 있다1,2,3,4,5.

생리적 손상 외에도

근이영양증4,6,7 및 암 유발 악액질8에서 근육 손상과 재생이 발생하며,

노화 과정에서는 이 과정이 결함을 보인다9,10.

기존 연구에 따르면

근섬유 사멸 직후 근육 재생이 시작되며,

이로 인해 면역 세포가 근육으로 유입된다.

이러한 침윤 세포는

세포 잔해 제거 및 근육 생성 촉진 능력(즉, 위성 근육 줄기세포의 융합을 통한 새로운 근섬유 형성)을 통해

근육 재생에 필수적이다2,3,11,12,13.

근육 재생 과정에서는

호중구, 단핵구, 대식세포, T 세포 등 여러 차례의 면역 세포 모집이 발생한다14.

호중구는 근육 재생 초기 단계에 모집되며,

이후 단계에서는 M1/M2 대식세포가 뒤따른다2,3,11,12,13.

호중구와 M1 대식세포는 염증에 기여하는 반면,

M2 대식세포는 항염증 기능을 가진다2,3,11,12,13,15.

호중구와 대식세포의 부분적 고갈은

근육 재생을 저해한다16,17,18,19,

이는 침입하는 면역 세포가

근육 수복에 실제로 필요함을 시사한다.

중요한 점은 최적의 근육 재생에는 강력하지만

일시적인 다양한 면역 세포 집단의 동원, 그리고

염증 상태에서 항염증 상태로의 전환이 필요하다는 것이다2,3,11,12,13.

결과적으로 지속적인 염증은

근육병증 및 노화 관련 근육 수리 결핍의 원인이다20,21,22,23,24.

While macrophages have been extensively investigated19,25,26, relatively less is known about the role of neutrophils in tissue repair27,28,29,30 and muscle regeneration2,3,11,12,13,18. Upon injury, neutrophils rapidly invade the damaged muscle, where they remove cellular debris and secrete inflammatory factors that recruit monocytes and macrophages2,3,11,12,13,18. Eventually, neutrophils move back to the circulation starting from 24 h after injury2,3,11,12,13. Altogether, neutrophils are pivotal for the phagocytosis of necrotic material and for stimulating the homing of other inflammatory cells2,3,11,12,13,18. However, persistent infiltration of neutrophils into muscles exacerbates inflammation, causes further damage, and delays the subsequent steps of muscle regeneration6,31,32,33. Consequently, it was found that depleting neutrophils starting from 24 h after injury accelerates the transition to the subsequent phases of muscle regeneration and hence improves muscle repair34. Therefore, a rapid and robust but transient recruitment of neutrophils is necessary for efficient muscle regeneration. Despite their importance, the mechanisms responsible for the timely recruitment of neutrophils to injured muscles are incompletely understood. Previous studies have found that necrotic myofibers activate the complement system and muscle-resident mast cells and neutrophils, which in turn release pro-inflammatory factors that promote the massive recruitment of additional neutrophils from the circulation11,32,33,35,36. However, it remains undetermined whether neutrophil recruitment from the bloodstream is simply dependent on cues released from injured myofibers and muscle-resident cells or whether other signals are also necessary.

Platelets are anucleated blood cells derived from megakaryocytes and known for their role in hemostasis37,38,39,40.

대식세포는 광범위하게 연구되어 왔지만19,25,26,

호중구의 조직 회복27,28,29,30 및 근육 재생2,3,11,12,13,18에서의 역할은

상대적으로 덜 알려져 있다.

손상 시 호중구는

손상된 근육으로 신속히 침투하여 세포 잔해를 제거하고

단핵구 및 대식세포를 모집하는 염증 인자를 분비한다2,3,11,12,13,18.

결국 호중구는

손상 후 24시간부터 순환계로 재이동하기 시작한다2,3,11,12,13.

종합하면,

호중구는

괴사 물질의 식균 작용과

다른 염증 세포의 귀소(homing)를 자극하는 데

핵심적 역할을 합니다2,3,11,12,13,18.

그러나

근육으로의 지속적인 호중구 침윤은 염증을 악화시키고

추가 손상을 유발하며

근육 재생의 후속 단계를 지연시킵니다6,31,32,33.

결과적으로,

손상 후 24시간부터 호중구를 제거하면

근육 재생의 후속 단계로의 전환이 가속화되어

근육 수리가 개선된다는 사실이 밝혀졌다34.

따라서

효율적인 근육 재생을 위해서는 신속하고 강력하지만

일시적인 호중구 모집이 필요하다.

그 중요성에도 불구하고,

손상된 근육으로 호중구가 적시에 모집되는 메커니즘은 완전히 이해되지 않았다.

기존 연구에 따르면

괴사성 근섬유는

보체 시스템과 근육 상주 비만세포 및 호중구를 활성화시키며,

이들이 차례로 순환계로부터 추가 호중구의 대량 유입을 촉진하는

전염증성 인자를 방출한다11,32,33,35,36.

그러나

혈류로부터의 호중구 모집이 단순히 손상된 근섬유와 근육 상주 세포에서 방출되는 신호에 의존하는지,

아니면 다른 신호도 필요한지는 아직 밝혀지지 않았다.

혈소판은

거핵세포에서 유래한 핵이 없는 혈액 세포로,

지혈 작용에 관여하는 것으로 알려져 있다37,38,39,40.

Specifically, platelets are activated by many stimuli associated with blood vessel injury and are responsible for clot formation in the immediate phase that follows injury41,42. Upon activation, platelets release the contents of alpha and dense secretory granules, which include cytokines, growth factors, and metabolites43,44,45,46. Platelet-secreted factors have been found to contribute to many processes beyond coagulation47,48,49. Interestingly, several studies have explored the therapeutic use of the platelet-rich plasma (PRP), i.e., the component of the blood devoid of white and red blood cells but rich in platelets and platelet-secreted factors (releasate). In particular, injection of the PRP has been proposed to boost wound healing and regeneration in a number of tissues50,51,52,53,54,55,56,57,58, including skeletal muscle59,60,61,62,63,64. However, it remains largely undetermined whether platelets are necessary for skeletal muscle regeneration beyond their role in hemostasis, and whether platelet-secreted factors are required for any step of muscle repair.

Here, we show that platelet-secreted chemokines guide the early steps of muscle repair by recruiting neutrophils to injured muscles. Perturbation of this early step of regeneration compromises the re-establishment of myofiber size and muscle strength in mice. Altogether, these findings indicate a key role for platelet-derived signals in initiating skeletal muscle regeneration.

특히 혈소판은

혈관 손상과 관련된 다양한 자극에 의해 활성화되며,

손상 직후 단계에서 혈전 형성을 담당한다41,42.

활성화된 혈소판은

알파 분비 소체와 고밀도 분비 소체의 내용물을 방출하는데,

여기에는 사이토카인, 성장 인자 및 대사 산물이 포함된다43,44,45,46.

혈소판 분비 인자들은

응고 작용을 넘어 다양한 과정에 기여하는 것으로 밝혀졌습니다47,48,49.

흥미롭게도,

여러 연구에서 혈소판이 풍부한 혈장(PRP)의 치료적 활용을 탐구해 왔습니다.

즉, 백혈구와 적혈구를 제외한 혈액 성분으로,

혈소판과 혈소판 분비 인자(릴리즈에이트)가 풍부한 것입니다.

특히,

PRP 주사는

골격근59,60,61,62,63,64를 비롯한 여러 조직에서

상처 치유 및 재생을 촉진하는 것으로 제안되었습니다50,51,52,53,54,55,56,57,58.

그러나

혈소판이 지혈 작용을 넘어 골격근 재생에 필수적인지,

그리고 혈소판 분비 인자가 근육 회복의

어느 단계에 필요한지는 아직 대부분 밝혀지지 않았다.

본 연구에서는

혈소판 분비 케모카인이 손상된 근육으로 호중구를 유인함으로써

근육 회복의 초기 단계를 유도함을 보여줍니다.

재생 과정의 이 초기 단계를 방해하면

생쥐에서 근섬유 크기와 근력의 회복이 저해됩니다.

종합적으로, 이러한 결과는

골격근 재생의 시작 단계에서

혈소판 유래 신호가 핵심적인 역할을 함을 시사합니다.

Results

Platelets are detected in skeletal muscles upon injury and this can be prevented via antibody-based platelet depletion

To start to investigate whether platelets contribute to skeletal muscle regeneration, we have first examined via immunostaining whether platelets are present in skeletal muscles upon injury. To this purpose, we have utilized an experimental model of injury induced by intramuscular injection of cardiotoxin (CTX) into the tibialis anterior (TA) skeletal muscle of mice65,66,67, (Fig. 1a). Whereas no platelets are found in uninjured muscles, platelet aggregates were prominent at day 1, were present at day 7, and eventually declined at day 14 after CTX injection (Fig. 1b–d). Co-immunostaining with the endothelial cell marker PECAM-1 indicated that platelet aggregates, which have pro-hemostatic effects68,69,70, are located primarily within blood vessels of injured skeletal muscles (Supplementary Fig. 1).

결과

골격근 손상 시 혈소판이 검출되며, 항체 기반 혈소판 제거를 통해 이를 방지할 수 있음

혈소판이 골격근 재생에 기여하는지 조사하기 위해,

우리는 먼저 손상 시 골격근에 혈소판이 존재하는지 면역염색을 통해 확인했습니다.

이를 위해 우리는

쥐의 전경골근(TA) 골격근에 심장독소(CTX)를 근육 내 주사하여

유발한 손상 실험 모델을 활용했습니다65,66,67, (그림 1a).

손상되지 않은 근육에서는 혈소판이 발견되지 않았으나,

CTX 주사 후 1일차에는 혈소판 응집체가 두드러지게 관찰되었고,

7일차에도 존재했으며,

결국 14일차에는 감소하는 양상을 보였다(그림 1b–d).

내피 세포 마커인 PECAM-1과의 공동 면역염색을 통해,

지혈 촉진 효과가 있는 혈소판 응집체68,69,70이

주로 손상된 골격근의 혈관 내에 위치함을 확인했습니다(보충 그림 1).

Fig. 1: Platelet thrombi are found in injured muscles and are prevented via antibody-based platelet depletion.

a Experimental strategy to assess the role of platelets in skeletal muscle regeneration. The i.v. injection of a platelet-depleting antibody is done 2 h before muscle injury and is repeated at day 4 after injury. Control mice are injected with an IgG control antibody. Muscle injury is induced via the injection of cardiotoxin (CTX) into a tibialis anterior (TA) muscle whereas the contralateral TA muscle is mock-injected with PBS. TA muscles are retrieved at day 1, 7, and 14 for further analyses. b–d Immunostaining of TA muscles from control and platelet-depleted mice, either injured via the injection of cardiotoxin (CTX) or uninjured (mock-injected with PBS). WGA (red) provides an outline of myofibers whereas platelet aggregates (green) of different sizes are detected with anti-GP1bβ antibodies. Platelet thrombi are found in injured muscles at day 1 and day 7 from CTX-induced injury but their presence is minimal at day 14 from injury and they are not detected in uninjured TA muscles. Antibody-based platelet depletion results in the lack of platelets aggregates in injured muscles, indicating that this strategy is effective for testing the role of platelets in muscle regeneration. The graph displays the mean ±SD with n = 5 (from 5 control independent mice) and n = 4 (from 4 platelet-depleted independent mice) biologically independent uninjured muscles; n = 7 biologically independent CTX-injured muscles obtained from n = 7 independent control mice at day 7 after injury; and n = 10 biologically independent CTX-injured muscles obtained from n = 10 independent mice for each of the other timepoints and conditions analyzed; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (two-way ANOVA with Tukey post hoc test); &&&P < 0.001 (two-way ANOVA with Sidak post hoc test) refers to the comparison of muscles from control versus platelet-depleted mice at a given timepoint of regeneration. Source data are provided in the Source data file.

그림 1: 손상된 근육에서 혈소판 혈전이 발견되며, 항체 기반 혈소판 고갈을 통해 이를 방지할 수 있다.

a 골격근 재생에서 혈소판의 역할을 평가하기 위한 실험 전략. 혈소판 고갈 항체를 근육 손상 2시간 전에 정맥 주사하고, 손상 후 4일째에 다시 주사한다. 대조군 마우스에는 IgG 대조군 항체를 주사한다. 근육 손상은 전경골근(TA)에 카디오톡신(CTX)을 주사하여 유도하며, 대측 TA 근육에는 PBS를 모의 주사한다. TA 근육은 추가 분석을 위해 1일, 7일, 14일째에 회수한다. b–d 대조군 및 혈소판 제거 마우스의 TA 근육 면역염색 결과(심장독소(CTX) 주사로 손상된 경우 또는 손상되지 않은 경우(PBS 모의 주사)). WGA(적색)는 근섬유의 윤곽을 보여주며, 항-GP1bβ 항체로 다양한 크기의 혈소판 응집체(녹색)가 검출된다. CTX 유발 손상 후 1일 및 7일차 손상 근육에서 혈소판 혈전이 관찰되나, 손상 14일차에는 그 존재가 최소화되며 손상되지 않은 TA 근육에서는 검출되지 않는다. 항체 기반 혈소판 제거는 손상 근육 내 혈소판 응집체 부재를 초래하여, 이 전략이 근육 재생에서 혈소판의 역할을 검증하는 데 효과적임을 시사한다. 그래프는 생물학적으로 독립적인 비손상 근육의 평균 ±표준편차(SD)를 표시하며, n = 5(대조군 독립 마우스 5마리) 및 n = 4(혈소판 제거 독립 마우스 4마리)로 구성됨; n = 7: 생물학적으로 독립적인 대조군 생쥐 n = 7에서 부상 7일 후 채취한 생물학적으로 독립적인 CTX 손상 근육; n = 10: 분석된 다른 시점 및 조건 각각에 대해 생물학적으로 독립적인 생쥐 n = 10에서 채취한 생물학적으로 독립적인 CTX 손상 근육; *P＜0.05, **P＜0.01, ***P＜0.001, ****P＜0.0001 (Tukey 사후 검정을 포함한 양방향 분산분석); &&&P<0.001 (Sidak 사후 검정을 포함한 양방향 분산분석)은 특정 재생 시점에서 대조군과 혈소판 제거 마우스의 근육 비교를 나타냅니다. 원본 데이터는 원본 데이터 파일에 제공됩니다.

Full size image

To determine the functional significance of platelet recruitment to injured muscles, we next examined skeletal muscle regeneration in the absence of platelets. To this purpose, 2 h before intramuscular CTX injection, mice were injected via the tail vein with a platelet-depleting antibody, which has been previously shown to reliably ablate platelets within 30 min from injection and for up to 3-4 days from injection71,72,73,74. Additionally, a second dose of platelet-depleting antibody was injected at day 4 following CTX injection (Fig. 1a). Control mice were treated in the same way but injected with a mock IgG antibody, as previously done in other studies that utilized these tools71,72,73,74 (Fig. 1a). As expected, tail-vein injection of the platelet-depleting antibody resulted in the consequent lack of platelets in injured TA muscles at day 1 and 7 after CTX injection (Fig. 1b–d). Altogether, this indicates that platelets localize to skeletal muscle early after injury, and that this is prevented by antibody-based platelet depletion.

전체 크기 이미지

손상된 근육으로의 혈소판 유입의 기능적 중요성을 확인하기 위해,

우리는 혈소판이 없는 상태에서의 골격근 재생 과정을 조사했습니다.

이를 위해, 근육 내 CTX 주사 2시간 전에, 쥐에게

꼬리 정맥을 통해 혈소판 제거 항체를 주사했다.

이 항체는 주사 후 30분 이내에 혈소판을 확실히 제거하며,

주사 후 최대 3-4일 동안 효과가 지속되는 것으로 이전에 입증된 바 있다71,72,73,74.

또한, CTX 주사 후 4일째에 혈소판 제거 항체를 두 번째로 주사했다(그림 1a). 대조군 마우스는 동일한 방식으로 처리되었으나, 이전 연구들에서 사용된 것과 동일한 방식으로 모의 IgG 항체를 주입하였다71,72,73,74 (그림 1a). 예상대로, 혈소판 제거 항체의 꼬리 정맥 주입은 CTX 주사 후 1일 및 7일째에 손상된 TA 근육에서 혈소판 결핍을 초래하였다 (그림 1b–d). 종합하면, 이는 혈소판이 손상 후 조기에 골격근으로 국소화되며, 항체 기반 혈소판 고갈에 의해 이 과정이 차단됨을 시사한다.

Platelets are necessary for neutrophil recruitment to injured skeletal muscles

Muscle regeneration relies on the recruitment of immune cells (e.g., neutrophils and macrophages) that are tasked with the removal of cellular debris and the subsequent promotion of myogenesis2,3,4,11. Neutrophils localize to injured muscles in the early phase of regeneration and set the stage for the subsequent invasion of regenerating muscles by macrophages12,13,31. However, the mechanisms responsible for the recruitment of neutrophils from the bloodstream are incompletely understood.

Because platelet aggregates are found in skeletal muscles early after injury (Fig. 1a–d), we hypothesize that they may regulate the recruitment of neutrophils to injured muscles. To test this model, hematoxylin/eosin (H&E) staining of TA muscle sections was utilized to determine the impact of antibody-mediated platelet depletion on muscle regeneration. Whereas infiltrates of immune cells were clearly seen at day 1 after injury in mock-treated mice, such invading immune cells were nearly absent in TA muscles obtained from platelet-depleted mice (Fig. 2a, b). To further test these findings, muscle sections were stained with known neutrophil markers, MMP-9 and Ly6G. As expected based on previous studies2,3,11,12,13, neutrophils were rare in uninjured muscles but abundant at day 1 after CTX injection. Platelet depletion significantly reduced neutrophil recruitment to muscles at day 1 after CTX injection whereas no substantial effects were seen at day 7 and 14 after injury (Fig. 2a–e), timepoints at which there is minimal presence of neutrophils in muscle2,3,11,12,13. Altogether, these findings indicate that platelet depletion impairs neutrophil recruitment to injured skeletal muscles.

손상된 골격근으로의 호중구 모집에 혈소판이 필수적이다

근육 재생은

세포 잔해 제거 및 후속 근육 생성 촉진2,3,4,11을 담당하는

면역 세포(예: 호중구 및 대식세포)의 모집에 의존한다.

호중구는 재생 초기 단계에 손상된 근육에 국소화되어,

이후 재생 중인 근육으로 대식세포가 침입할 수 있는 기반을 마련한다12,13,31.

그러나

혈류에서 호중구를 모집하는 메커니즘은 완전히 이해되지 않았다.

손상 직후 골격근에서 혈소판 응집체가 발견되기 때문에(그림 1a–d),

우리는 혈소판 응집체가 손상된 근육으로의 호중구 모집을 조절할 수 있다고 가정합니다.

이 모델을 검증하기 위해,

TA 근육 절편의 헤마톡실린/에오신(H&E) 염색을 활용하여

항체 매개 혈소판 고갈이 근육 재생에 미치는 영향을 확인하였다.

대조군 처리 마우스에서는 손상 후 1일째에 면역 세포 침윤이 명확히 관찰된 반면, 혈소판 고갈 마우스의 TA 근육에서는 이러한 침입 면역 세포가 거의 존재하지 않았다(그림 2a, b). 이러한 결과를 추가로 검증하기 위해 근육 절편을 알려진 호중구 표지자인 MMP-9 및 Ly6G로 염색하였다. 기존 연구2,3,11,12,13에 기반한 예상대로, 호중구는 손상되지 않은 근육에서는 드물었으나 CTX 주사 후 1일차에는 풍부하게 관찰되었다. 혈소판 제거는 CTX 주사 후 1일째 근육으로의 호중구 유입을 현저히 감소시켰으나, 손상 후 7일 및 14일째에는 유의미한 효과가 관찰되지 않았다(그림 2a–e). 이 시점에는 근육 내 호중구 존재가 최소화된다2,3,11,12,13.

종합하면, 이러한 결과는

혈소판 제거가 손상된 골격근으로의 호중구 유입을 저해함을

시사한다.

Fig. 2: Neutrophil infiltration in injured muscles is impeded by platelet depletion whereas late-phase macrophage infiltration is increased.

a, b H&E staining of TA muscles from control and platelet-depleted mice at day 1, 7, and 14 from cardiotoxin (CTX)-induced injury. In agreement with previous studies, immune infiltration is found at day 1 after CTX in control TA muscles but it is largely reduced in the TA muscles from platelet-depleted mice. At later stages, the process of muscle regeneration is impaired, as indicated by the overall lower size of myofibers and by the ultrastructural defects of TA muscles that are found at day 14. There are no noticeable changes in uninjured muscles from platelet-depleted versus control mice. c Immunostaining of TA muscles from control and platelet-depleted mice for neutrophil markers, i.e., MMP9 (red) and Ly6G (white). Myofiber boundaries are identified with immunostaining for anti-Laminin antibodies (green) whereas nuclei are identified by DAPI (blue). d–e Neutrophil infiltration in injured muscles occurs predominantly at day 1 from injury, and it is significantly reduced by platelet depletion. Similar results are found with the quantitation of both Ly6G and MMP9 immunostaining. f Quantitation of macrophages infiltrating the muscle, as defined with anti-F4/80 antibodies. Macrophage infiltration is predominant at day 7 from CTX-mediated injury and it is exacerbated by platelet depletion. In d-f, the graphs display the mean ±SD with n = 5 (from 5 control independent mice) and n = 4 (from 4 platelet-depleted independent mice) biologically independent uninjured muscles; n = 9 biologically independent CTX-injured muscles obtained from n = 9 independent control mice at day 7 after injury; and n = 10 biologically independent CTX-injured muscles obtained from n = 10 independent mice for each of the other timepoints and conditions analyzed; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (two-way ANOVA with Tukey post hoc test); &&&&P < 0.0001 (two-way ANOVA with Sidak post hoc test) refers to the comparison of muscles from control versus platelet-depleted mice at a given timepoint of regeneration. Source data are provided in the Source data file.

Full size image

Normally, staggered waves of immune cells are recruited to skeletal muscle upon injury to remove cell debris, to promote the recruitment of subsequent populations of immune cells, and to promote myogenesis2,3,11,12,13. On this basis, we next examined the impact of platelet depletion on the infiltration of macrophages, which are recruited to injured muscles in later phases of muscle regeneration. As expected2,3,11,12,13, immunostaining of muscle sections revealed that macrophages are more abundant in skeletal muscles at day 7 compared to uninjured muscles and to day 1 after injury (Fig. 2f and Supplementary Fig. 2). Interestingly, platelet depletion significantly increased overall macrophage infiltration into regenerating muscles at day 7 from injury (Fig. 2f and Supplementary Fig. 2), and this was due to higher levels of both M1 and M2 macrophages (Supplementary Fig. 3). These findings might be explained by the fact that reduced neutrophil recruitment due to platelet depletion may impede the removal of cell debris in the early phases of regeneration, which may exacerbate inflammation and lead to a higher macrophage recruitment at later stages.

그림 2: 손상된 근육에서의 호중구 침윤은 혈소판 고갈에 의해 저해되는 반면, 후기 단계 대식세포 침윤은 증가한다.

a, b 심장독소(CTX) 유발 손상 후 1일, 7일, 14일째 대조군 및 혈소판 고갈 마우스의 TA 근육 H&E 염색. 기존 연구와 일치하게, 대조군 TA 근육에서는 CTX 투여 후 1일째 면역 세포 침윤이 관찰되나, 혈소판 고갈 마우스의 TA 근육에서는 크게 감소한다. 후기 단계에서는 근섬유의 전반적인 크기 감소와 14일차에 관찰된 TA 근육의 초구조적 결함으로 나타난 바와 같이 근육 재생 과정이 손상된다. 혈소판이 제거된 마우스와 대조군 마우스의 손상되지 않은 근육에는 눈에 띄는 변화가 없습니다. c 대조군 및 혈소판이 제거된 마우스의 TA 근육에 대한 호중구 마커, 즉 MMP9(빨간색) 및 Ly6G(흰색)의 면역염색. 근섬유 경계는 항-라미닌 항체(녹색) 면역염색으로 확인되며, 핵은 DAPI(파란색)로 확인됩니다. d–e 손상된 근육의 호중구 침윤은 주로 손상 후 1일째에 발생하며, 혈소판 고갈에 의해 현저히 감소합니다. Ly6G 및 MMP9 면역염색 정량화에서도 유사한 결과가 관찰됨. f 항-F4/80 항체로 정의된 근육 침윤 대식세포의 정량화. 대식세포 침윤은 CTX 매개 손상 7일차에 가장 두드러지며, 혈소판 제거 시 악화됨. d-f의 그래프는 생물학적으로 독립적인 비손상 근육 n = 5(대조군 독립 마우스 5마리) 및 n = 4(혈소판 제거 독립 마우스 4마리)의 평균 ±표준편차를 나타냄; n = 9: 생물학적으로 독립적인 대조군 생쥐 n = 9마리에서 손상 7일 후 채취한 생물학적으로 독립적인 CTX 손상 근육; *P＜0.05, **P＜0.01, ***P＜0.001, ****P＜0.0001 (Tukey 사후 검정을 포함한 양방향 분산분석); &&&&P<0.0001 (Sidak 사후 검정을 포함한 양방향 분산분석)은 재생의 특정 시점에서 대조군과 혈소판 제거 마우스의 근육 비교를 나타냅니다. 원본 데이터는 원본 데이터 파일에 제공됩니다.

전체 크기 이미지

일반적으로 골격근 손상 시 면역 세포는

단계적으로 파도처럼 유입되어

세포 잔해를 제거하고,

후속 면역 세포 집단의 유입을 촉진하며,

근육 생성을 촉진합니다2,3,11,12,13.

이를 바탕으로, 우리는 다음으로

근육 재생 후기 단계에 손상된 근육으로 유입되는

대식세포의 침윤에 대한 혈소판 제거의 영향을 조사했습니다.

예상대로2,3,11,12,13, 근육 절편의 면역염색 결과

대식세포는 손상되지 않은 근육 및 손상 후 1일차에 비해 7일차 골격근에서

더 풍부하게 존재하는 것으로 나타났다(그림 2f 및 보충 그림 2).

흥미롭게도,

혈소판 고갈은 손상 7일째 재생 중인 근육으로의 전체 대식세포 침윤을 유의미하게 증가시켰으며(그림 2f 및 보충 그림 2),

이는 M1 및 M2 대식세포 모두의 수준이 높아진 데 기인했습니다(보충 그림 3).

이러한 결과는 혈소판 고갈로 인한 호중구 모집 감소가

재생 초기 단계에서 세포 잔해 제거를 방해할 수 있으며,

이는 염증을 악화시켜 후기 단계에서 더 많은 대식세포 모집으로 이어질 수 있다는 사실로 설명될 수 있다.

Platelets are necessary for the growth of newly-formed myofibers and for the optimal regeneration of injured muscles

Optimal muscle regeneration depends on waves of immune cell recruitment to injured muscles and their interaction with regenerating myofibers2,3,11,12,13. We have found that platelet depletion impairs the timely recruitment of neutrophils to injured muscles (Fig. 2).

On this basis, we next examined muscles at 1, 7, and 14 days after CTX injection to test whether there are consequent effects on skeletal muscle regeneration. Staining with phalloidin and anti-Laminin antibodies revealed that muscle regeneration is impeded by platelet depletion, as indicated by the lower size of myofibers of TA muscles from platelet-depleted mice at day 14 post-injury (Fig. 3a–c). Specifically, measurement of the myofiber size via estimation of the Feret’s minimal diameter indicates that platelet depletion reduces the size of myofibers at day 14 but not at day 7 post-injury. Collective analysis of all myofibers derived from the TA muscles of each group further indicates that platelet depletion overall decreases the range of myofiber sizes at day 14 post-injury (Fig. 3d).

혈소판은 새로 형성된 근섬유의 성장과 손상된 근육의 최적 재생에 필수적이다

최적의 근육 재생은

손상된 근육으로의 면역 세포 모집 파동과 재생 중인 근섬유와의 상호작용에 달려 있다2,3,11,12,13.

우리는 혈소판 고갈이

손상된 근육으로의 호중구 적시 모집을 저해한다는 사실을 발견했다(그림 2).

이를 바탕으로,

골격근 재생에 미치는 영향 여부를 확인하기 위해

CTX 주사 후 1일, 7일, 14일째 근육을 관찰하였다.

팔로이딘 및 항-라미닌 항체 염색을 통해,

혈소판 고갈이 근육 재생을 방해한다는 사실이 밝혀졌습니다.

이는 손상 14일 후 혈소판 고갈 마우스의 TA 근육에서

근섬유 크기가 더 작다는 점으로 확인되었습니다(그림 3a–c).

구체적으로,

페레의 최소 직경 추정을 통한 근섬유 크기 측정은 혈소판 고갈이 손상 후 14일째에는

근섬유 크기를 감소시키지만 7일째에는 그렇지 않음을 나타낸다.

각 군의 TA 근육에서 유래한 모든 근섬유를 종합적으로 분석한 결과, 혈소판 고갈은 손상 후 14일째에 근섬유 크기 범위를 전반적으로 감소시키는 것으로 나타났다(그림 3d).

Fig. 3: Platelet depletion impedes the growth of myofibers during regeneration.

a Immunostaining of TA muscles from control and platelet-depleted mice at day 1, 7, and 14 from cardiotoxin (CTX)-induced injury with phalloidin (to detect F-actin; red) and with anti-Laminin antibodies (green) to detect the myofiber boundaries. b, c Quantitation of myofiber sizes (as estimated with the Feret’s minimal diameter) based on anti-Laminin and phalloidin staining. Myofibers detected at day 1 largely consist of necrotic myofibers whereas myofibers found at day 7–14 are new myofibers resulting from de novo myogenesis. There are no significant changes in the size of myofibers found at day 7 in the muscles from platelet-depleted versus control mice, suggesting that platelet depletion does not impair myogenesis (see also Supplementary Fig. 4). However, myofiber size is significantly reduced at day 14 from CTX-induced injury in the muscles from platelet-depleted versus control mice. The graphs display the mean ±SD. In b, n = 4 (from 4 control independent mice) and n = 6 (from 6 platelet-depleted independent mice) biologically independent CTX-injured muscles at day 7 after injury; and n = 10 biologically independent CTX-injured muscles obtained from n = 10 independent mice for each of the other timepoints and conditions analyzed. In c, n = 8 (from 8 control independent mice) biologically independent CTX-injured muscles at day 7 after injury; and n = 10 biologically independent CTX-injured muscles obtained from n = 10 independent mice for each of the other timepoints and conditions analyzed. **P < 0.01, ***P < 0.001, ****P < 0.0001 (two-way ANOVA with Tukey post hoc test); &P < 0.05 (two-way ANOVA with Sidak post hoc test) refers to the comparison of muscles from control versus platelet-depleted mice at day 14 of regeneration. d Gaussian plots that show the size range of all myofibers sourced from all TA muscles here analyzed, stained for F-actin. There are minimal changes in myofiber size (Feret’s minimal diameter) at day 7 whereas platelet depletion leads to a significant reduction in myofiber size at day 14 from CTX injection. Source data are provided in the Source data file.

그림 3: 혈소판 고갈은 재생 과정 중 근섬유의 성장을 방해한다.

a 심장독소(CTX) 유발 손상 후 1일, 7일, 14일째 대조군 및 혈소판 고갈 마우스의 TA 근육 면역염색. 팔로이딘(F-액틴 검출용; 적색) 및 라미닌 항체(근섬유 경계 검출용; 녹색)를 사용함. b, c 라미닌 및 팔로이딘 염색을 기반으로 한 근섬유 크기 정량화(페레의 최소 직경으로 추정). 1일차에 검출된 근섬유는 대부분 괴사된 근섬유인 반면, 7일차 –14일차에 발견된 근섬유는 신규 근생성(de novo myogenesis)에 의한 새로운 근섬유이다. 혈소판 제거 마우스와 대조군 마우스의 근육에서 7일차에 발견된 근섬유의 크기에는 유의미한 차이가 없었으며, 이는 혈소판 제거가 근생성을 저해하지 않음을 시사한다(보충 그림 4 참조). 그러나 CTX 유발 손상 후 14일째 혈소판 제거 마우스의 근육에서는 대조군 대비 근섬유 크기가 현저히 감소하였다. 그래프는 평균 ±표준편차를 나타낸다. b에서는 손상 후 7일째 생물학적으로 독립적인 CTX 손상 근육 n = 4(대조군 독립 마우스 4마리) 및 n = 6(혈소판 제거 독립 마우스 6마리)를 표시한다; 그리고 분석된 다른 시점 및 조건 각각에 대해 독립적인 생물학적 개체 n = 10마리에서 얻은 생물학적으로 독립적인 CTX 손상 근육 n = 10개. c에서는 손상 후 7일째 생물학적으로 독립적인 CTX 손상 근육 n = 8개(대조군 독립 개체 8마리에서 유래); 그리고 분석된 다른 시점 및 조건 각각에 대해 독립적인 생물학적 개체 n = 10마리에서 얻은 생물학적으로 독립적인 CTX 손상 근육 n = 10개. **P＜0.01, ***P＜0.001, ****P＜0.0001 (Tukey 사후 검정을 포함한 양방향 분산분석); &P<0.05 (Sidak 사후 검정을 통한 이원 분산분석)은 재생 14일차에 대조군 대 혈소판 제거 마우스 근육 간 비교를 나타냄. d F-액틴 염색을 통해 분석된 모든 TA 근육에서 유래한 모든 근섬유의 크기 범위를 보여주는 가우시안 플롯. 7일차에는 근섬유 크기(Feret 최소 직경)에 최소한의 변화만 관찰되나, 혈소판 제거는 CTX 주사 후 14일차에 근섬유 크기의 유의미한 감소를 초래한다. 원본 데이터는 원본 데이터 파일에 제공된다.

Full size image

The decline in myofiber size due to platelet depletion may depend on an impediment of myogenesis, i.e., the formation of new myofibers from satellite muscle stem cells. To examine whether myogenesis is impacted by platelet depletion, we measured the abundance of eMHC-positive myofibers. Normally, eMHC (embryonic myosin heavy chain) is expressed during muscle development and disappears after birth but it is re-expressed during myogenesis associated with muscle regeneration75,76,77. In agreement with this knowledge, there are many eMHC-positive myofibers at 7 days after cardiotoxin-induced injury, coincident with myogenesis, but their abundance declines at day 14 post-injury (Supplementary Fig. 4), a timepoint at which myogenesis and muscle regeneration are largely resolved. Similar percentages of eMHC-positive tissue areas are found in regenerating skeletal muscles from control and platelet-depleted mice (Supplementary Fig. 4), suggesting that platelet depletion does not impair myogenesis. However, our finding that myofiber size is reduced 14 days post-injury in platelet-depleted mice (Fig. 3) suggests that the lack of platelets impairs the growth of newly-formed myofibers.

전체 크기 이미지

혈소판 제거로 인한 근섬유 크기 감소는

근육 형성, 즉 위성 근육 줄기세포로부터 새로운 근섬유가 형성되는 과정의 저해에 기인할 수 있다.

혈소판 제거가 근육 형성에 영향을 미치는지 확인하기 위해

eMHC 양성 근섬유의 풍부도를 측정하였다.

정상적으로

eMHC(배아형 미오신 중쇄)는 근육 발달 중에 발현되며 출생 후 사라지지만,

근육 재생과 관련된 근육 형성 과정에서 재발현됩니다75,76,77.

이러한 지식과 일치하게, 심장독소 유발 손상 7일 후에는 근육 형성과 동시에 많은 eMHC 양성 근섬유가 존재하지만, 이들의 풍부도는 손상 후 14일째(보조 그림 4)에 감소합니다. 이 시점은 근육 형성과 근육 재생이 대부분 해결된 시점입니다. 대조군과 혈소판 고갈 마우스의 재생 중인 골격근에서 유사한 비율의 eMHC 양성 조직 영역이 발견됩니다(보조 그림 4). (보충 그림 4). 이 시점에는 근육형성과 근육 재생이 대부분 완료된다. 대조군과 혈소판 제거 마우스의 재생 중인 골격근에서 eMHC 양성 조직 영역의 비율은 유사하게 관찰된다(보충 그림 4). 이는 혈소판 제거가 근육형성을 저해하지 않음을 시사한다. 그러나 혈소판 제거 마우스에서 손상 14일 후 근섬유 크기가 감소한다는 우리의 발견(그림 3)은 혈소판 결핍이 새로 형성된 근섬유의 성장을 저해함을 시사한다.

Cytokine profiling of regenerating skeletal muscles indicates that platelet depletion impairs neutrophil chemotactic signaling, neo-angiogenesis, and myofiber growth

Upon activation, platelets release the content of secretory granules, which consists of cytokines, growth factors, and metabolites43,44,45,46. Previous studies have found that platelet-secreted factors can contribute to tissue repair47,48,49. On this basis, platelet depletion may impact muscle regeneration because of decreased levels of platelet-secreted factors and/or modulation of cytokine production by muscle cells and infiltrating immune cells.

To test this hypothesis, we have utilized Quantibody (Quantitative Multiplex ELISA) arrays to profile the protein levels of 640 mouse cytokines in extracts from TA skeletal muscles obtained from mice with or without antibody-mediated platelet depletion at 1, 7, and 14 days after CTX-induced muscle injury. In addition, we also profiled the levels of cytokines in uninjured muscles 7 days after platelet depletion.

As expected, the levels of several cytokines were modulated at different timepoints of muscle regeneration but the highest difference in the profile of secreted factors between platelet-depleted versus controls was found at day 1 after regeneration (Fig. 4a, b). GO term analysis of significantly-regulated cytokines in platelet-depleted versus control TA muscles indicates that several categories of cytokines are collectively regulated at different timepoints of muscle regeneration in a platelet-dependent manner (Fig. 4c). At day 1 after injury, platelet depletion leads to a remarkable decline in the levels of cytokines that promote neutrophil chemotaxis. At this early timepoint, there is also a significant decline in the levels of VEGF (Supplementary Fig. 6a), a potent inducer of blood vessel formation which can be secreted by platelets as well as by neutrophils during tissue regeneration30,78,79,80,81,82. Coincident with the reduction in VEGF levels, we find that post-injury muscles from platelet-depleted mice have defects in neo-angiogenesis, as indicated by immunostaining for PECAM1 (Supplementary Fig. 6b, c).

재생 중인 골격근의 사이토카인 프로파일링은 혈소판 고갈이 호중구 화학유인 신호전달, 신생혈관 형성 및 근섬유 성장을 저해함을 나타낸다.

활성화 시 혈소판은

사이토카인, 성장 인자 및 대사 산물로 구성된 분비 소체 내용물을 방출한다43,44,45,46.

기존 연구에 따르면 혈소판 분비 인자는

조직 복구에 기여할 수 있다47,48,49.

이러한 근거에 따르면,

혈소판 고갈은 혈소판 분비 인자의 감소 및/또는 근육 세포와 침윤 면역 세포에 의한 사이토카인 생산 조절로 인해

근육 재생에 영향을 미칠 수 있다.

이 가설을 검증하기 위해, 우리는 퀀티바디(Quantibody, 정량적 다중 ELISA) 어레이를 활용하여 CTX로 유발된 근 손상 후 1일, 7일, 14일 시점에서 항체 매개 혈소판 고갈이 있는 마우스와 없는 마우스의 TA 골격근 추출물에서 640종의 마우스 사이토카인 단백질 수준을 프로파일링하였다. 또한, 혈소판 고갈 7일 후 손상되지 않은 근육의 사이토카인 수준도 프로파일링하였다.

예상대로 여러 사이토카인 수준은 근육 재생의 서로 다른 시점에서 조절되었으나, 재생 후 1일차에 혈소판 제거 대조군 대비 분비 인자 프로파일에서 가장 큰 차이가 관찰되었다(그림 4a, b). . 혈소판 제거 대조군과 대조군 TA 근육에서 유의하게 조절된 사이토카인의 GO 용어 분석은 근육 재생의 서로 다른 시점에서 여러 범주의 사이토카인이 혈소판 의존적 방식으로 집단적으로 조절됨을 나타낸다(그림 4c). 손상 후 1일째, 혈소판 제거는 호중구 화학유인을 촉진하는 사이토카인 수치의 현저한 감소를 초래한다. 이 초기 시점에서는 혈관신생의 강력한 유도제인 VEGF(보충그림 6a) 수치도 유의미하게 감소하는데, 이는 조직 재생 중 혈소판과 호중구 모두에 의해 분비될 수 있다30,78,79, 80,81,82. VEGF 수준 감소와 동시에, 혈소판 제거 마우스의 손상 후 근육에서 PECAM1 면역염색 결과로 나타난 신생혈관형성 결함이 관찰되었다(보충 그림 6b, c).

Fig. 4: Platelet depletion reduces the intramuscular levels of neutrophil chemoattractants in the early phase of muscle regeneration.

a Principal Component Analysis (PCA) of 640 cytokines profiled with Quantibody (Quantitative Multiplex ELISA) arrays from TA muscle homogenates obtained from mice with or without platelet depletion at 1, 7, and 14 days from CTX-induced injury. Uninjured muscles after 7 days from platelet depletion were also analyzed. b Heatmap of 522 cytokines that are prominently regulated (based on the average z-scores) during muscle regeneration and/or in response to platelet depletion. c Cytokine categories that are collectively regulated at different timepoints of muscle regeneration in a platelet-dependent manner include cytokines that promote neutrophil chemotaxis (day 1 from injury) and the inflammatory response (day 7–14 from injury). See also Supplementary Fig. 5. d Chemokines that promote neutrophil chemotaxis peak at day 1 after injury but their levels are reduced by platelet depletion. The graphs display the mean ±SD with n = 4 biologically independent muscles from 4 independent mice for each timepoint and condition; *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired two-tailed t test) refer to the comparison of muscles from control versus platelet-depleted mice at a given timepoint. e In vitro neutrophil chemotaxis assays with recombinant versions of platelet-secreted chemokines. CXCL5 and CXCL7 have the strongest chemoattractant activity. The graphs display the mean ±SD with n = 17 (buffer), n = 18 (rCXCL7), n = 18 (rCXCL5), n = 6 (rCXCL4) biologically independent samples; ***P < 0.001 (one-way ANOVA with Tukey post hoc test), ns = not significant. f Consistent with the cytokine array data in d, ELISA assays indicate that the total levels of CXCL7 increase in skeletal muscle upon injury. The graph displays the mean ±SD with n = 11 biologically independent samples; ***P < 0.0001 (unpaired two-tailed t test). g Additional ELISA assays with antibodies specific for inactive CXCL7 (i.e., which has not been proteolytically processed) indicate a decrease in inactive CXCL7 in injured muscles. The graph displays the mean ±SD with n = 11 biologically independent samples; ***P = 0.0004 (unpaired two-tailed t test). Together, these data indicate that the surge in total CXCL7 observed in injured muscles largely consists of proteolytically-cleaved (and hence active) CXCL7. Source data are provided in the Source data file.

그림 4: 혈소판 제거는 근육 재생 초기 단계에서 근육 내 호중구 화학유인물질 수준을 감소시킨다.

a CTX 유발 손상 후 1, 7, 14일째 혈소판 제거 유무에 따른 TA 근육 균질물에서 Quantibody(정량적 다중 ELISA) 어레이로 프로파일링한 640개 사이토카인의 주성분 분석(PCA). 혈소판 제거 후 7일째의 무손상 근육도 분석함. b 근육 재생 과정 및/또는 혈소판 고갈에 반응하여 현저히 조절되는(평균 z-점수 기준) 522개 사이토카인의 히트맵. c 근육 재생의 서로 다른 시점에서 혈소판 의존적 방식으로 집단적으로 조절되는 사이토카인 범주에는 중성구 화학유인(손상 후 1일차) 및 염증 반응(손상 후 7~14일차)을 촉진하는 사이토카인이 포함된다. 보충 그림 5도 참조하십시오. d 호중구 화학유도를 촉진하는 케모카인은 손상 후 1일째에 최고치를 보이지만, 혈소판 고갈 시 그 수준이 감소합니다. 그래프는 각 시점 및 조건에 대해 4마리의 독립적인 생쥐에서 유래한 생물학적으로 독립적인 근육 4개(n = 4)의 평균 ±표준편차를 표시합니다; *P＜0.05, **P＜0.01, ***P＜0.001 (짝을 이루지 않은 양측 t 검정)은 특정 시점에서 대조군 대 혈소판 제거 마우스의 근육 비교를 나타냅니다. e 재조합 혈소판 분비 케모카인을 이용한 체외 중성구 화학주행 분석. CXCL5와 CXCL7이 가장 강력한 화학유인 활성을 나타냄. 그래프는 생물학적으로 독립적인 샘플 n = 17 (버퍼), n = 18 (rCXCL7), n = 18 (rCXCL5), n = 6 (rCXCL4)의 평균 ±표준편차(SD)를 표시함; ***P<0.001 (Tukey 사후 검정을 포함한 일원 분산 분석), ns = 유의미하지 않음. f d의 사이토카인 어레이 데이터와 일관되게, ELISA 분석은 손상 시 골격근에서 CXCL7의 총 수준이 증가함을 나타낸다. 그래프는 생물학적으로 독립적인 n=11개 샘플의 평균 ±표준편차(SD)를 표시함; ***P<0.0001 (짝을 이루지 않은 양측 t 검정). g 비활성 CXCL7(즉, 단백질 분해 처리되지 않은)에 특이적인 항체를 사용한 추가 ELISA 분석은 손상된 근육에서 비활성 CXCL7이 감소함을 나타냄. 그래프는 생물학적으로 독립적인 n = 11개 샘플의 평균 ±표준편차를 표시함; ***P = 0.0004 (짝을 이루지 않은 양측 t 검정). 종합적으로, 이 데이터는 손상된 근육에서 관찰된 총 CXCL7의 급증이 주로 단백질 분해적으로 절단된(따라서 활성인) CXCL7로 구성됨을 나타냅니다. 원본 데이터는 원본 데이터 파일에 제공됩니다.

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In later phases of muscle regeneration (day 7 and 14), platelet depletion resulted in increased levels of cytokines associated with the inflammatory response (Fig. 4c), which is suggestive of excessive and persistent inflammation that may impair regeneration and myofiber growth83. In particular, tumor necrosis factor ligands (TNFs) were significantly upregulated in muscles from platelet-depleted mice at day 7 (Supplementary Fig. 5). Although transient TNF signaling promotes myogenesis during muscle regeneration84,85,86,87, activation of this pathway has been found to stunt myofiber growth and to induce myofiber atrophy88,89,90,91. Therefore, the upregulation of TNF ligands in the regenerating muscles of platelet-depleted mice likely contributes to the reduced myofiber size that is found in these muscles post-injury (Fig. 3).

We have found that platelet depletion reduces the infiltration of neutrophils in regenerating skeletal muscles. On this basis, the lack of platelets may decrease neutrophil recruitment to injured muscles because of decreased levels of platelet-secreted cytokines necessary for neutrophil chemotaxis. Consistent with this hypothesis, the levels of several chemokines reported to promote neutrophil chemotaxis92,93 were reduced (Fig. 4d). Specifically, the protein levels of CXCL1, CXCL2, CXCL4/PF4, CXCL5, and CXCL7/PPBP increase at day 1 after injury, coincident with the phase of neutrophil recruitment to injured skeletal muscle. However, such increase in the protein levels of chemokines that promote neutrophil recruitment is impeded by platelet depletion. Quantitatively, CXCL4/PF4 and CXCL7/PPBP have concentrations ~1000x higher than CXCL1, CXCL2, and CXCL5 (Fig. 4d), suggesting that these chemokines may have a predominant role.

CXCL4/PF4, CXCL5, and CXCL7/PPBP are CXC chemokines that are nearly exclusively expressed in megakaryocytes and platelets94,95,96,97,98. Beyond this cell type-specificity of expression‎, platelets are the most abundant source of bioactive CXCL4/PF4, CXCL5, and CXCL7/PPBP because these chemokines are stored in α-granules and can be immediately released at high (μM) concentrations upon platelet activation94,95,96,97,98.

Although these chemokines have been previously reported to recruit neutrophils, in vitro migration assays with recombinant versions of these chemokines indicate that CXCL5 and CXCL7 have the strongest chemotactic functions whereas recombinant CXCL4/PF4 has limited capacity to recruit neutrophils (Fig. 4e). Because CXCL7 activity depends on its N-terminal proteolytic processing, we also examined whether the surge in CXCL7 protein levels observed in injured muscles consists of proteolytically-cleaved (and hence active) CXCL7 and found it to be the case (Fig. 4f, g). On this basis, it is plausible that these platelet-specific chemokines are responsible for the recruitment of neutrophils to injured muscles, and that their reduced levels upon platelet depletion is the cause of defects in neutrophil infiltration.

Neutrophil infiltration in regenerating muscles is reduced in Cxcl7KO mice

To test the hypothesis that platelet-specific chemokines regulate muscle regeneration by impacting neutrophil recruitment, we have utilized Cxcl7/Ppbp knockout (Cxcl7ko) mice99, which have been found to exhibit normal platelet numbers and hemostatic functions (i.e., the platelet thrombotic response occurs normally upon activation)99. These mice have been obtained via the targeted deletion of the Cxcl7/Ppbp coding sequence99 and, as expected, this leads to consequent loss of the plasma protein levels of CXCL7 (Fig. 5a). Although not directly targeted by the deletion of the Cxcl7/Ppbp coding sequence, the plasma levels of CXCL4/PF4 and CXCL5 also decline (Fig. 5a), presumably because these chemokines are encoded by genes that are located in adjacent loci and hence their expression‎ is affected by deletion of the Cxcl7/Ppbp coding sequence99. On the other hand, the levels of CXCL1, which is encoded by a gene located in another genomic region, are not affected (Fig. 5a). Therefore, the Cxcl7ko mice that lack CXCL4, CXCL5, and CXCL7 provide a useful system to test the requirement of platelet-specific chemokines in neutrophil recruitment to injured muscles.

전체 크기 이미지

근육 재생 후기 단계(7일 및 14일)에서 혈소판 고갈은 염증 반응과 연관된 사이토카인 수치 증가를 초래했습니다(그림 4c). 이는 재생 및 근섬유 성장을 저해할 수 있는 과도하고 지속적인 염증을 시사합니다83. 특히, 혈소판 고갈 마우스의 근육에서 종양괴사인자 리간드(TNFs)가 7일차에 유의미하게 상향 조절되었습니다 (보충 그림 5). 일시적인 TNF 신호전달은 근육 재생 중 근육형성을 촉진하지만84,85,86,87, 이 경로의 활성화는 근섬유 성장을 저해하고 근섬유 위축을 유발하는 것으로 밝혀졌다88,89,90,91. 따라서 혈소판이 제거된 생쥐의 재생 중인 근육에서 TNF 리간드의 발현 증가가 손상 후 이 근육에서 관찰되는 근섬유 크기 감소에 기여할 가능성이 높다(그림 3).

우리는 혈소판 고갈이 재생 중인 골격근으로의 호중구 침윤을 감소시킨다는 사실을 발견했습니다. 이를 바탕으로, 혈소판 부족은 호중구 화학유도에 필요한 혈소판 분비 사이토카인 수치의 감소로 인해 손상된 근육으로의 호중구 모집을 감소시킬 수 있습니다. 이 가설과 일치하게, 호중구 화학유도를 촉진하는 것으로 보고된 여러 케모카인 수준이 감소했습니다(그림 4d). 구체적으로, CXCL1, CXCL2, CXCL4/PF4, CXCL5 및 CXCL7/PPBP의 단백질 수준은 손상 후 1일째에 증가하며, 이는 손상된 골격근으로의 호중구 모집 단계와 일치한다. 그러나 호중구 모집을 촉진하는 케모카인의 단백질 수준 증가가 혈소판 제거에 의해 저해된다. 정량적으로, CXCL4/PF4와 CXCL7/PPBP의 농도는 CXCL1, CXCL2 및 CXCL5보다 약 1000배 높습니다(그림 4d). 이는 이러한 케모카인이 주요한 역할을 할 수 있음을 시사합니다.

CXCL4/PF4, CXCL5 및 CXCL7/PPBP는 거핵세포와 혈소판에서 거의 독점적으로 발현되는 CXC 케모카인이다94,95,96,97,98. 이러한 세포 유형 특이적 발현 외에도, 혈소판은 생리활성 CXCL4/PF4, CXCL5 및 CXCL7/PPBP의 가장 풍부한 공급원입니다. 이는 해당 케모카인들이 α-과립에 저장되어 있으며, 혈소판 활성화 시 즉시 높은 농도(μM)로 방출될 수 있기 때문입니다94,95,96,97,98.

이러한 케모카인들은 이전에 호중구를 모집하는 것으로 보고되었지만, 재조합 버전의 이러한 케모카인을 사용한 시험관 내 이동 분석에 따르면 CXCL5와 CXCL7이 가장 강력한 화학주성 기능을 나타내는 반면, 재조합 CXCL4/PF4는 호중구를 모집하는 능력이 제한적입니다(그림 4e). CXCL7의 활성은 N-말단 단백질 분해 처리 과정에 의존하므로, 손상된 근육에서 관찰된 CXCL7 단백질 농도 급증이 단백질 분해 처리된(따라서 활성 상태인) CXCL7로 구성되는지 조사한 결과, 실제로 그러한 것으로 확인되었다(그림 4f, g). 이러한 근거를 바탕으로, 이러한 혈소판 특이적 케모카인들은 손상된 근육으로의 호중구 모집을 담당하며, 혈소판 고갈 시 이들의 감소된 수준이 호중구 침윤 결손의 원인이라는 가설이 타당하다.

재생 중인 근육에서의 호중구 침윤은 Cxcl7KO 마우스에서 감소한다

혈소판 특이적 케모카인이 호중구 모집에 영향을 미쳐 근육 재생을 조절한다는 가설을 검증하기 위해, 우리는 Cxcl7/Ppbp 녹아웃(Cxcl7ko) 마우스99를 활용하였다. 이 마우스는 정상적인 혈소판 수와 지혈 기능(즉, 활성화 시 혈소판 혈전 반응이 정상적으로 발생함)을 나타내는 것으로 확인되었다99. 이 마우스는 Cxcl7/Ppbp 코딩 서열의 표적 삭제99를 통해 얻었으며, 예상대로 이로 인해 CXCL7의 혈장 단백질 수준이 감소한다(그림 5a). Cxcl7/Ppbp 코딩 서열 삭제에 직접적으로 영향을 받지 않음에도 불구하고, CXCL4/PF4 및 CXCL5의 혈장 수준도 감소한다(그림 5a). 이는 아마도 이들 케모카인이 인접한 유전자좌에 위치한 유전자들에 의해 암호화되기 때문에, Cxcl7/Ppbp 코딩 서열 삭제로 인해 그들의 발현이 영향을 받기 때문일 것이다99. 반면, 다른 게놈 영역에 위치한 유전자에 의해 암호화되는 CXCL1의 수준은 영향을 받지 않는다(그림 5a). 따라서 CXCL4, CXCL5 및 CXCL7이 결핍된 Cxcl7ko 마우스는 손상된 근육으로의 호중구 모집에 혈소판 특이적 케모카인의 필요성을 검증하는 유용한 모델 시스템을 제공한다.

Fig. 5: Neutrophil infiltration in injured muscles is impeded by Cxcl7ko platelets.

a Plasma levels of the neutrophil chemoattractant CXCL7 are reduced in Cxcl7ko (Cxcl7-/-) mice. CXCL5 and CXCL4 (encoded by adjacent genes) are also reduced whereas CXCL1 is not affected. The graphs display the mean ± SD with n = 4 (CXCL7) and n = 5 (CXCL1, CXCL4, CXCL5) biologically independent samples from n = 4 and n = 5 independent mice, respectively; *P < 0.05 and **P < 0.01 (unpaired two-tailed t test). b, c H&E staining of TA muscles from control and Cxcl7ko mice at day 1, 7, and 14 from cardiotoxin (CTX) injection, and uninjured. Immune infiltration at day 1 after CTX-mediated injury is reduced in the muscles from Cxcl7ko mice. Ultrastructural defects can be seen at day 14 post-injury in muscles from Cxcl7ko versus control mice. d Immunostaining of muscles from control and Cxcl7ko mice for neutrophil markers, i.e., Ly6G (purple). Myofiber boundaries are identified with immunostaining for anti-Laminin antibodies (green) whereas nuclei are stained with DAPI (blue). e Intramuscular neutrophil infiltration at day 1 from injury is significantly reduced in Cxcl7ko mice. Similar results are found with both Ly6G and MMP9 immunostaining. The graphs display the mean ±SD with n = 5 biologically independent samples for each group and condition from n = 5 independent mice; **P < 0.01, ***P < 0.001, ****P < 0.0001 (two-way ANOVA with Tukey post hoc test); &P < 0.05 and &&&P < 0.001 (two-way ANOVA with Sidak post hoc test) refer to the comparison of CTX-injured WT and Cxcl7ko at a given timepoint of regeneration. f Normally, platelets (green) are found in association with neutrophils (red) in injured muscles. Although lack of CXCL7 decreases neutrophil recruitment, the area of platelet thrombi that is found in injured muscles of WT and Cxcl7ko mice is similar, indicating that defective neutrophil recruitment does not result from lower platelet number or aggregation in Cxcl7ko mice, consistent with previous studies that have found that platelet numbers and hemostatic functions are not impaired in these mice99. The graph displays the mean ±SD with n = 5 biologically independent samples for each group and condition from n = 5 independent mice; ***P < 0.001 (two-way ANOVA with Tukey post hoc test). Source data are provided in the Source Data file.

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For these studies, we have used similar approaches as for the analysis of muscles from platelet-depleted mice (Fig. 2). Specifically, hematoxylin/eosin (H&E) staining of TA muscle sections from Cxcl7ko mice was utilized to determine the impact on muscle regeneration of loss of the platelet-specific chemokines CXCL4/5/7. Infiltration of immune cells in injured muscles was found, as expected, at day 1 after CTX injection in control mice but this was greatly reduced in the TA muscles from Cxcl7ko mice (Fig. 5b, c). Analyses of samples from 7 and 14 days after CTX injection further revealed that muscle regeneration is impeded by CXCL4/5/7 loss, as indicated by irregularities in the tissue ultrastructure (Fig. 5b, c).

Immune cells invading the muscle at day 1 after CTX-induced injury primarily consist of neutrophils2,3,11,12,13. To test whether decreased recruitment of immune cells due to CXCL4/5/7 loss primarily results from reduced neutrophil recruitment, we have monitored via immunostaining the levels of the neutrophil markers MMP-9 and Ly6G (Fig. 5d, e). Co-staining with platelet markers revealed that neutrophils can be found in close association with platelet thrombi (Fig. 5f). Loss of CXCL4/5/7 significantly reduced neutrophil recruitment to muscles at day 1 after CTX injection, compared to controls (Fig. 5d, e) and this was not due to changes in the amount of platelet thrombi detected in injured muscle (Fig. 5f). Altogether, these findings indicate that Cxcl7 knockout impairs neutrophil recruitment to injured skeletal muscles.

Automated image analysis reveals a similar pattern of defective muscle regeneration in response to platelet depletion and Cxcl7ko

We next used image analysis based on machine learning to determine whether injured (CTX-injected) TA muscles from platelet-depleted and Cxcl7ko mice display similar defects in regeneration compared to control muscles. Automated analysis of H&E images reliably identified muscle-infiltrating immune cells, myofibers, and centrally-nucleated myofibers (Supplementary Fig. 7a), which are indicative of newly formed myofibers resulting from de novo myogenesis2,3. As expected, there were substantially no centrally-nucleated myofibers and minimal immune infiltration in uninjured (PBS-injected) TA muscles.

Compared to wild-type injured controls, TA muscles from platelet-depleted and Cxcl7ko mice displayed similar defects in immune cell infiltration at day 1 after injury (Supplementary Fig. 7b, d). These findings are consistent with the identification of defective neutrophil infiltration by immunostaining at this early stage of regeneration in the muscles from platelet-depleted and Cxcl7ko mice (Figs. 2–5).

The number of centrally nucleated myofibers is maximal at day 7 and then declines by day 14, a late stage at which the muscle has largely regenerated its mass and strength3. There was a trend towards decreased levels of centrally nucleated myofibers in the muscles from platelet-depleted and Cxcl7ko mice but these values were not statistically significant (Supplementary Fig. 7c, e), suggesting that myogenesis is not substantially affected by platelet-derived CXCL4/5/7. Altogether, these automated image analyses indicate that defective immune infiltration at day 1 after injury is a major and shared feature of altered regeneration in the muscles of platelet-depleted and Cxcl7ko mice.

Defective neutrophil recruitment to injured muscles in Cxcl7ko mice leads to reduced myofiber size and muscle force production post-injury

We have found that platelets and the platelet-derived chemokines CXCL4, CXCL5, and CXCL7 guide neutrophil infiltration in the early steps of muscle regeneration (Figs. 1–5). In addition to cardiotoxin (CTX), intramuscular injection of glycerol is routinely used to induce skeletal muscle damage in mice80,81,100,101,102. Interestingly, glycerol elicits a stronger inflammatory response than CTX102, suggesting that glycerol-induced injury may constitute a useful setting to study the impact of platelet-induced signaling on immune cell recruitment to injured muscles. On this basis, we next tested whether defective platelet-derived chemokine signaling impairs muscle regeneration also after glycerol-induced injury. To this purpose, we examined the TA muscles from WT and Cxcl7ko mice 14 days after glycerol-induced injury, a post-injury stage in which muscle regeneration is largely completed2,3,32. In agreement with this model, all mice displayed recovery of the TA muscle mass and even post-injury muscle hypertrophy (Fig. 6a).

Fig. 6: Myofiber size and force production are reduced in post-injury muscles from Cxcl7ko mice.

Analysis of TA muscles after 14 days from glycerol-induced injury. a TA muscle weight normalized by the tibia bone length indicates that there is post-injury hypertrophy, although this is not significant for Cxcl7ko mice. b There is no significant difference in the twitch force of uninjured and post-injury muscles from WT mice, indicating that regeneration has recovered muscle function. However, post-injury muscles from Cxcl7ko mice are significantly weaker compared to uninjured muscles. c Similar deficits in muscle force are found in pre-fatigue muscles from Cxcl7ko mice, whereas there are no differences post-fatigue. In a–c, the graphs display the mean ±SD with n = 12 (WT) and n = 11 (Cxcl7ko) biologically independent samples from n = 12 and n = 11 independent mice, respectively; *P < 0.05 (two-way ANOVA with Tukey post hoc test), ns = not significant. d Immunostaining of uninjured and post-injury muscles from control and Cxcl7ko mice with antibodies for myosin heavy chain isoforms to detect type 2a myofibers (green), type 2x myofibers (black), and type 2b myofibers (red). Defects in regeneration (such as space in-between myofibers) are found in post-injury muscles from Cxcl7ko mice compared to post-injury controls. e–g Gaussian plots indicate an overall reduced size (Feret’s minimal diameter) of type 2a (e) and type 2x (f) myofibers in post-injury muscles from Cxcl7ko mice, compared to post-injury muscles from WT mice and uninjured controls. h Quantitation of myofiber sizes based on the average values obtained from the individual muscles in a group. There is a significant decline in the size of myofibers in post-injury muscles from Cxcl7ko mice whereas myofiber size differences between uninjured and post-injury WT muscles are not significant. i There is an overall similar myofiber type composition of uninjured and post-injury TA muscles. j Myofiber number similarly increases in post-injury versus uninjured muscles from WT and Cxcl7ko. In h–j, the graphs display the mean ±SD with n = 12 (WT) and n = 11 (Cxcl7ko) biologically independent samples from n = 12 and n = 11 independent mice, respectively; *P < 0.05, **P < 0.01, ***P < 0.001, ns = not significant (two-way ANOVA with Tukey post hoc test). Source data are provided in the Source data file.

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To examine the functional consequences of defective TA muscle regeneration in Cxcl7ko mice, we next measured the twitch force (i.e., physiological, spontaneous-like force) generated by WT and Cxcl7ko mice 14 days after injury. Consistent with the overall completion of regeneration at this stage2,3,32, there was no significant difference in the twitch force of post-injury versus uninjured WT muscles (Fig. 6b). However, TA muscles from Cxcl7ko mice displayed significant reduction in the twitch force compared to uninjured controls, indicating that muscle regeneration is incomplete, presumably due to impairment of signaling by platelet-specific CXCL4/5/7 chemokines (Fig. 6b). Analysis of muscle fatigability, which is based on a series of maximal (tetanic) stimulations of TA muscles, revealed no difference post-fatigue but confirmed that post-injury TA muscles from Cxcl7ko mice but not from WT mice display reduced force production in the pre-fatigue state (Fig. 6c).

To understand the mechanistic basis of decreased muscle force in Cxcl7ko mice post-injury, we analyzed the size of distinct myofiber types in TA muscles from WT and Cxcl7ko mice, at 14 days post-injury or uninjured (Fig. 6d). These histological analyses highlight that regeneration is defective in post-injury muscles from Cxcl7ko mice, as indicated by the presence of debris and empty spaces in-between the myofibers, compared to post-injury muscles from isogenic control mice (Fig. 6d). Moreover, the Gaussian plots (representing all myofibers sourced from all TA muscles in each group) indicate that the Feret’s minimal diameter of type 2a and type 2x myofibers is reduced in Cxcl7ko mice at 14 days post-injury (Fig. 6e, f) whereas the size of type 2b myofibers is not affected compared to post-injury WT muscles (Fig. 6g).

The analysis of the myofiber size across individual TA muscles led to overall similar conclusions: there was no significant difference in the size of type 2a, 2x, and 2b myofibers when comparing uninjured versus post-injury WT muscles (Fig. 6h). However, there was a significant decline in the size of myofibers in post-injury versus uninjured muscles from Cxcl7ko mice (Fig. 6h). Such decline in myofiber size was not accompanied by any major changes in the relative proportion of different myofiber types that compose the TA muscle (Fig. 6i). Moreover, there was a higher myofiber number in both WT and Cxcl7ko post-injury muscles (Fig. 6j), consistent with previous studies that have examined TA muscle regeneration2,3,32, although such increase was significant only for WT muscles (Fig. 6j). We also analyzed the intramuscular fat infiltration, which occurs in post-injury muscles in particular after glycerol-induced injury80,81,100,102, but found no difference when comparing muscles from WT versus Cxcl7ko mice (Supplementary Fig. 8).

Altogether, these studies indicate that physiological force production by skeletal muscles from Cxcl7ko mice is impaired post-injury due to reduced myofiber size, and that platelet-derived CXCL4/5/7 chemokine signaling is an early step of muscle regeneration that is key for the re-establishment of skeletal muscle function.

Neutrophil depletion impairs myofiber growth and neo-angiogenesis during muscle regeneration

Previous studies have found that neutrophils are necessary for muscle regeneration2,3,18. Here, we have found that platelet depletion and Cxcl7 knockout impair neutrophil recruitment to injured muscles and that this in turn impedes myofiber growth and force production in regenerating muscles (Figs. 2–6). To test whether neutrophils are indeed necessary for muscle regeneration, we have utilized anti-Gr1 and anti-Ly6G antibodies to deplete neutrophils and compared these mice to IgG-treated controls. For each of these mice, one leg was injured with glycerol whereas the contralateral leg was injected with PBS (control).

Histological analyses of tibialis anterior muscles at 10 days post-injury indicate that myofiber size is reduced in the muscles of neutrophil-depleted versus mock-treated mice whereas the uninjured muscles are not affected by neutrophil depletion (Fig. 7a). Gaussian curves (obtained from the cumulative analysis of all myofibers in a group of muscles) indicate that the size of type 2a, 2x, and 2b myofibers is reduced in post-injury muscles upon neutrophil depletion compared to IgG control treatments whereas there is no effect of neutrophil depletion on the size of myofibers in the contralateral uninjured muscles (Fig. 7b–d). Analysis of the average myofiber size for each mouse indicates that neutrophil depletion significantly reduces the size of type 2x (P = 0.019) and type 2b (P = 0.0036) myofibers in post-injury muscles compared to mock-treated controls and that the size of type 2a myofibers is also reduced, albeit with P = 0.0570 (Fig. 7e). There are no major effects on the myofiber type distribution, apart for an increase in the percentage of type 2x myofibers which occurs in both neutrophil-depleted and controls (Fig. 7f). Lastly, these histological analyses indicate that there is an overall increase in the number of myofibers in post-injury muscles from both neutrophil-depleted and control mice (Fig. 7g). Altogether, these findings indicate that neutrophil depletion impairs myofiber growth during muscle regeneration.

Fig. 7: Neutrophil depletion impairs skeletal muscle regeneration.

Analysis of TA muscles after 10 days from glycerol-induced injury. a Immunostaining for myosin heavy chain isoforms was utilized to detect type 2a (green), type 2x (black), and type 2b myofibers (red): these histological analyses indicate defective muscle regeneration in mice with neutrophil depletion. b–d Gaussian plots indicate that neutrophil depletion impairs the growth of newly formed myofibers in post-injury muscles whereas there is no effect of neutrophil depletion on myofiber size in contralateral uninjured muscles. e Quantitation of myofiber sizes (Feret’s minimal diameter) based on the average values obtained from the individual muscles in a group. There is an overall significant decline in the size of type 2x and 2b myofibers in post-injury muscles from neutrophil-depleted mice compared to post-injury muscles from mock-treated mice. f There is an overall similar myofiber type composition of uninjured and post-injury TA muscles. However, post-injury muscles (both from neutrophil-depleted and mock-treated mice) display higher levels of type 2x myofibers. g The number of type 2x and 2b myofibers increases in post-injury versus uninjured muscles and the number of type 2b myofibers is significantly higher in the muscles from neutrophil-depleted mice. In e–g, the graphs display the mean ±SD with n = 6 (from 6 independent control mice) and n = 4 (from 4 independent neutrophil-depleted mice) biologically independent muscles; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (two-way ANOVA with Tukey post hoc test), ns = not significant. h The normalized twitch force of post-injury muscles from neutrophil-depleted mice is reduced compared to that of post-injury muscles from mock-treated mice. i Similar deficits in muscle force production are found for the normalized tetanic force of muscles from neutrophil-depleted versus mock-treated mice. Neutrophil depletion does not impact twitch and tetanic force production by uninjured muscles (h, i). In h, i, the graphs display the mean ± SD with n = 6 (from 6 independent control mice) and n = 4 (from 4 independent neutrophil-depleted mice) biologically independent muscles; *P < 0.05, ****P < 0.0001 (two-way ANOVA with Sidak post hoc test). Source data are provided in the Source data file.

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In addition to removing debris and promoting the organized recruitment of other immune cell types, neutrophils promote tissue regeneration also via the secretion of many signaling factors, which include regulators of angiogenesis such as the vascular endothelial growth factor (VEGF)30,82,103,104,105,106,107. Moreover, neutrophils express high levels of MMP9 and other metalloproteases that release additional VEGF bound to the extracellular matrix, further enhancing VEGF bioavailability28,82. On this basis, we have examined the abundance of capillaries (based on PECAM-1 staining) and found that neutrophil depletion significantly reduces neo-angiogenesis in post-injury muscles compared to mock-treated controls (Supplementary Fig. 9), as also observed upon platelet depletion (Supplementary Fig. 6). Moreover, analysis of fat infiltration (identified by perilipin-1 staining) indicates a trend towards an increase (Supplementary Fig. 9). Coincident with the decline in myofiber size and neo-angiogenesis, post-injury muscles from mice with neutrophil depletion produce a significantly lower normalized twitch and tetanic force compared to mock-treated controls whereas neutrophil depletion does not impact force production in the contralateral uninjured muscles (Fig. 7h, i).

Altogether, these studies indicate that the recruitment of neutrophils to injured muscles is necessary for optimal blood vessel formation, myofiber growth, and force production. On this basis, defective neutrophil recruitment appears to be a primary reason for the impairment of muscle regeneration in response to platelet depletion and knockout of platelet-derived Cxcl7.

Discussion

Skeletal muscle has the remarkable capacity to regenerate in response to injuries caused by many physiological and pathological insults1,2,3,4,5. The sequential recruitment of immune cell populations and the de novo formation of myofibers are key events in muscle regeneration that have been extensively studied2,3,11,12,13. However, less is known about the early steps of muscle repair and the consequences of their derangement on later phases of muscle regeneration and post-injury muscle function.

Platelets are among the first responders to tissue injuries108 but their role in muscle regeneration has not been explored. In addition to their classical roles in hemostasis, there is growing appreciation that platelets have important signaling functions109,110,111. For example, our previous work has found that platelets promote cancer cell metastasis via secretion of TGF-β and other pro-metastatic signaling factors that induce epithelial-to-mesenchymal transition and migration of cancer cells73,112,113,114. In this study, we have found that platelets guide the early steps of muscle regeneration by promoting the recruitment of neutrophils to injured muscles: platelet-specific chemokines (CXCL5 and CXCL794,95,96,97,98) are necessary for the infiltration of neutrophils into injured muscles, and impairment of such platelet-derived chemokine signaling impedes this process.

토론

골격근은

다양한 생리적 및 병리적 손상에 대한 반응으로 재생하는 놀라운 능력을 지닌다1,2,3,4,5.

면역 세포 집단의 순차적 모집과 근섬유의 신규 형성은

근육 재생의 핵심 사건으로 광범위하게 연구되어 왔다2,3,11,12,13.

그러나

근육 수복의 초기 단계와

그 장애가 근육 재생 후기 단계 및 손상 후 근육 기능에 미치는 영향에 대해서는 덜 알려져 있다.

혈소판은

조직 손상에 대한 최초 대응자 중 하나이지만108 

근육 재생에서의 역할은 탐구되지 않았다.

지혈에 대한 고전적인 역할 외에도,

혈소판이 중요한 신호 전달 기능을 가지고 있다는 인식이 점점 커지고 있다109,110,111.

예를 들어,

우리의 이전 연구에서는

혈소판이 TGF-β 및 기타 전이 촉진 신호 인자를 분비하여

암세포의 상피-중간엽 전이 및 이동을 유도함으로써

암세포 전이를 촉진한다는 사실을 발견했다73,112,113,114.

본 연구에서는

혈소판이 손상된 근육으로의 호중구 모집을 촉진함으로써

근육 재생의 초기 단계를 유도한다는 사실을 발견했습니다:

혈소판 특이적 케모카인(CXCL5 및 CXCL794,95,96,97,98)은

손상된 근육으로의 호중구 침투에 필수적이며,

이러한 혈소판 유래 케모카인 신호전달의 장애는 이 과정을 방해합니다.

Platelet localization and neutrophil recruitment to muscles is maximal within the first 24 h after injury and then declines in the subsequent days (Figs. 1 and 2). Likewise, the levels of the platelet-secreted neutrophil chemoattractants CXCL5 and CXCL7 are maximal at day 1 after injury (Fig. 4), indicating that platelet-initiated inter-cellular signaling occurs in the early steps of muscle regeneration, presumably immediately upon platelet activation and the formation of thrombi in injured muscles.

Interestingly, derangement of this early step of muscle regeneration has consequences at later stages. Specifically, platelet depletion and Cxcl7 knockout leads to the regeneration of muscles that have decreased myofiber size (Figs. 3 and 6), as also found with neutrophil depletion (Fig. 7). As estimated with the analysis of eMHC levels (Supplementary Fig. 4), myogenesis seems not be affected by platelet depletion, suggesting that the growth rather than the de novo formation of myofibers is impacted by platelets. However, this occurs in the late phases of muscle regeneration, a stage at which neutrophils have migrated out of the muscles and the levels of platelet-specific secreted factors (defined based on the platelet proteome115) detected in muscles are minimal (Fig. 4). There are however several cytokines and growth factors that are differentially modulated in the late phase of muscle regeneration when comparing platelet-depleted versus control mice. These include cytokines such as tumor necrosis factor ligands (Fig. 4 and Supplementary Fig. 5) that have been found to promote myogenesis but that are known inducers of myofiber atrophy during cancer cachexia and in other disease states that promote muscle wasting88,89,90,91. These factors are likely contributed by muscle-infiltrating cells other than platelets and neutrophils, such as macrophages, for which we find increased recruitment to injured muscles of platelet-depleted mice at day 7 from injury, compared to controls (Fig. 2f and Supplementary Figs. 2 and 3). Therefore, a plausible model is that defective platelet-derived chemokine signaling and reduced neutrophil recruitment to injured muscles in the early steps of muscle regeneration leads to unresolved tissue damage and consequent excessive inflammation and macrophage recruitment at later stages of regeneration, and that this stunts the growth of new myofibers because of the high levels of atrophic ligands. Moreover, we find that platelet depletion decreases VEGF levels in the early phase of muscle regeneration (Supplementary Fig. 6) and that neo-angiogenesis is impeded in post-injury muscles from mice with platelet depletion (Supplementary Fig. 6) and in mice with neutrophil depletion (Supplementary Fig. 9). Because neo-angiogenesis precedes myogenesis during regeneration116,117,118, defective neo-angiogenesis likely contributes to the impediment of myofiber growth observed upon depletion of neutrophils, platelets, and platelet-secreted chemokines.

손상 후 첫 24시간 이내에

혈소판의 국소화와 근육으로의 호중구 모집이 최대에 달한 후,

이후 며칠 동안 감소합니다(그림 1 및 2).

마찬가지로,

혈소판이 분비하는 호중구 화학유인물질인 CXCL5와 CXCL7의 수준은

손상 후 1일째에 최대치에 도달한다(그림 4).

이는 혈소판에 의해 시작되는 세포 간 신호전달이

근육 재생의 초기 단계,

즉 손상된 근육에서 혈소판 활성화 및 혈전 형성이 발생한 직후에 발생함을 시사한다.

흥미롭게도,

근육 재생의 이 초기 단계가 교란되면

후기 단계에 영향을 미칩니다.

구체적으로,

혈소판 고갈 및 Cxcl7 녹아웃은 근섬유 크기가 감소된 근육의 재생을 초래합니다(그림 3 및 6).

이는 호중구 고갈에서도 관찰된 바와 같습니다(그림 7).

eMHC 수준 분석(보충 그림 4)으로 추정된 바와 같이,

혈소판 고갈은 근육 형성에는 영향을 미치지 않는 것으로 보이며,

이는 근섬유의 신규 형성보다는 성장에 혈소판이 영향을 미친다는 것을 시사합니다.

그러나 이는 근육 재생 후기 단계에서 발생하며, 이 시점에는 호중구가 근육에서 이탈하고 근육에서 검출되는 혈소판 특이적 분비 인자(혈소판 프로테옴115을 기반으로 정의됨) 수준이 최소화된다(그림 4). 그러나 혈소판 고갈 마우스와 대조군 마우스를 비교할 때 근육 재생 후기 단계에서 차등적으로 조절되는 여러 사이토카인과 성장 인자들이 존재한다. 여기에는 종양 괴사 인자 리간드(그림 4 및 보충 그림 5)와 같은 사이토카인이 포함되며, 이는 근육 형성을 촉진하는 것으로 밝혀졌지만 암 악액질 및 기타 근육 소모를 촉진하는 질병 상태에서 근섬유 위축을 유발하는 것으로 알려져 있습니다88,89,90,91. 이러한 인자들은 혈소판 및 호중구 이외의 근육 침윤 세포, 예를 들어 대식세포에 의해 기여될 가능성이 높으며, 우리는 대조군에 비해 혈소판 고갈 마우스의 손상된 근육에 손상 7일째에 대식세포의 모집이 증가함을 발견했습니다(그림 2f 및 보충 그림 2 및 3).

따라서 타당한 모델은

근육 재생 초기 단계에서 혈소판 유래 케모카인 신호 전달 결함과

손상된 근육으로의 호중구 유입 감소가 해결되지 않은 조직 손상과

재생 후기 단계에서의 과도한 염증 및 대식세포 유입을 초래하고,

이로 인해 높은 수준의 위축성 리간드로 인해 새로운 근섬유의 성장이 저해된다는 것이다.

또한, 혈소판 고갈은

근육 재생 초기 단계에서 VEGF 수준을 감소시키고(보충 그림 6), 혈

소판 고갈 마우스(보충 그림 6)와 호중구 고갈 마우스(보충 그림 9)의 손상 후 근육에서

신생 혈관 형성이 방해받는다는 것을 발견했습니다.

신생 혈관 형성은 재생 과정에서 근육 형성에 선행하기 때문에116,117,118,

호중구, 혈소판 및 혈소판 분비 케모카인의 고갈 시 관찰되는 근섬유 성장 저해에는

신생 혈관 형성의 결함이 기여할 가능성이 높습니다.

In addition to promoting the removal of cellular debris and to setting the stage for the infiltration of other immune cells2,3,11,12,13,18, neutrophils also influence the metabolic capacity of skeletal muscle119. Specifically, it was previously found that neutrophils support muscle force production via secretion of IL-1β, which promotes muscle performance by priming exercise-dependent GLUT4 translocation and glucose metabolism119. We have found that impaired neutrophil recruitment due to CXCL7 loss results in decreased muscle force production post-injury. While this decreased muscle performance likely stems from reduced myofiber growth, it may also arise from derangement of neutrophil signaling to muscle satellite cells, and the consequent negative impact on muscle metabolism.

In addition to physiological repair, muscle regeneration is also altered in several diseases, such as muscular dystrophy, which is characterized by damage-regeneration cycles that ultimately lead to stem cell depletion and to the incapacity to repair skeletal muscles7. Specifically, the lack of dystrophin leads to membrane tears and a rise in Ca2+ levels which are ultimately responsible for myofiber necrosis in Duchenne muscular dystrophy7. Interestingly, previous studies have found that platelet adhesion and aggregation are defective in patients with Duchenne muscular dystrophy120,121,122. On this basis and considering our finding that platelets contribute to skeletal muscle regeneration, it is possible that impairment of platelet function contributes to the chronic inflammation and to the defective regeneration of dystrophic muscles, as we have observed in this study upon experimental depletion of platelets (Figs. 2 and 3). Future studies should address whether injection of functional platelets and/or recombinant CXCL7 can aid regeneration and reduce chronic inflammation in skeletal muscles of mouse models and patients with Duchenne muscular dystrophy. Likewise, injection of platelets or and/or recombinant CXCL7 may aid muscle regeneration by boosting neutrophil recruitment in the context of aging and age-related diseases such as diabetes, which are characterized by decreased regenerative capacity10,123,124,125.

Platelet-released chemokines may also help the regeneration of more severe injuries, such as volumetric muscle loss (VML), which consists in the quick loss of >20% muscle mass126,127. In this context, the signaling interactions between platelets and neutrophils and/or recombinant platelet-secreted chemokines (e.g., CXCL5/7) may help the regeneration of VML injuries by promoting the efficacy of currently-used interventions such as tissue and stem cell engraftment126,127,128. Conversely, limiting platelet-derived chemokine signaling could help prevent excessive neutrophil infiltration, which occurs in unresolved VML injuries and contributes to the impairment of muscle stem cell function129,130.

There is also growing interest in the possibility of using platelets for drug delivery131,132,133. For example, because of their capacity to interact with cancer cells, doxorubicin-loaded platelets have been used to selectively target cancer cells while reducing general doxorubicin toxicity134. On this basis and considering our finding that platelets are recruited to regenerating skeletal muscles, we propose that platelets might be employed as drug carriers to deliver pro-regeneration factors specifically to injured skeletal muscles.

Altogether, this study indicates that platelet-initiated chemokine signaling guides the early steps of muscle regeneration by promoting neutrophil recruitment and that this in turn impacts myofiber size and muscle strength post-injury (Fig. 8). We propose that platelet-derived chemokines may provide therapeutic opportunities for promoting muscle regeneration.

세포 잔해 제거 촉진 및 다른 면역 세포의 침투를 위한 기반 마련2,3,11,12,13,18 외에도,

호중구는 골격근의 대사 능력에도 영향을 미친다119.

구체적으로,

호중구는 IL-1β 분비를 통해 근력 생산을 지원하며,

이는 운동 의존적 GLUT4 전위 및 포도당 대사를 촉진하여 근육 성능을 향상시킨다는 사실이 이전에 밝혀졌다119.

우리는 CXCL7 결손으로 인한 호중구 모집 장애가

손상 후 근력 생산 감소를 초래함을 발견했다.

이러한 근 기능 저하는

근섬유 성장 감소에서 비롯될 가능성이 높지만,

호중구 신호전달이 근 위성세포에 미치는 영향 장애와 그에 따른 근 대사 부정적 영향에서도 기인할 수 있다.

생리적 회복 외에도 근육 재생은 근이영양증과 같은 여러 질환에서 변화한다. 근이영양증은 손상-재생 주기를 특징으로 하며, 이는 궁극적으로 줄기세포 고갈과 골격근 회복 능력 상실로 이어진다7. 특히 디스트로핀 결핍은 막 파열과 Ca2+ 농도 상승을 유발하며, 이는 뒤쉔형 근이영양증에서 근섬유 괴사의 근본 원인이다7. 흥미롭게도, 이전 연구들은 뒤쉔형 근이영양증 환자에서 혈소판 부착 및 응집이 결함이 있음을 발견했습니다120,121,122. 이를 바탕으로, 그리고 혈소판이 골격근 재생에 기여한다는 우리의 발견을 고려할 때, 혈소판 기능 장애가 만성 염증과 이영양성 근육의 결함 있는 재생에 기여할 가능성이 있습니다. 이는 본 연구에서 실험적으로 혈소판을 고갈시켰을 때 관찰된 바와 같습니다(그림 2 및 3). 향후 연구에서는 기능성 혈소판 및/또는 재조합 CXCL7 주사가 마우스 모델과 뒤쉔형 근이영양증 환자의 골격근에서 재생 촉진 및 만성 염증 감소에 기여할 수 있는지 규명해야 한다. 마찬가지로, 노화와 당뇨병과 같은 노화 관련 질환의 맥락에서 호중구 모집을 촉진함으로써 혈소판 또는 재조합 CXCL7의 주입이 근육 재생을 도울 수 있다. 이러한 질환들은 재생 능력의 감소를 특징으로 한다10,123,124,125.

혈소판에서 분비되는 케모카인은 또한 20% 이상의 근육량이 급격히 소실되는 체적성 근 손실(VML)과 같은 더 심각한 손상의 재생에도 도움이 될 수 있습니다126,127. 이러한 맥락에서, 혈소판과 호중구 및/또는 재조합 혈소판 분비 케모카인(예: CXCL5/7) 간의 신호 상호작용은 조직 및 줄기세포 이식126,127,128과 같은 현재 사용 중인 중재의 효능을 촉진함으로써 VML 손상의 재생을 도울 수 있습니다. 반대로, 혈소판 유래 케모카인 신호 전달을 제한하면 해결되지 않은 VML 손상에서 발생하는 과도한 호중구 침윤을 방지하는 데 도움이 될 수 있으며, 이는 근육 줄기 세포 기능 저하에 기여한다129,130.

또한 혈소판을 약물 전달에 활용할 가능성에 대한 관심도 증가하고 있다131,132,133. 예를 들어, 암세포와 상호작용할 수 있는 능력 덕분에 독소루비신을 탑재한 혈소판은 일반적인 독소루비신 독성을 줄이면서 암세포를 선택적으로 표적화하는 데 사용되어 왔다134. 이러한 근거와 재생 중인 골격근으로 혈소판이 모집된다는 우리의 연구 결과를 고려할 때, 혈소판을 약물 운반체로 활용하여 재생 촉진 인자를 손상된 골격근에 특이적으로 전달할 수 있을 것으로 제안한다.

종합하면, 본 연구는 혈소판에 의해 시작된 케모카인 신호전달이 호중구 모집을 촉진함으로써 근육 재생의 초기 단계를 유도하며, 이는 다시 손상 후 근섬유 크기와 근력에 영향을 미친다는 점을 시사한다(그림 8). 우리는 혈소판 유래 케모카인이 근육 재생 촉진을 위한 치료적 기회를 제공할 수 있다고 제안한다.

Fig. 8: Platelets promote skeletal muscle regeneration by guiding the recruitment of neutrophils to injured muscles via the platelet-released chemokine CXCL7.

In response to injury, platelets localize to and form thrombi in skeletal muscles and promote the recruitment of neutrophils via the release of platelet-specific chemokines (e.g., CXCL7) that are neutrophil chemoattractants. Neutrophil infiltration is known to promote muscle repair via the removal of cellular debris and by setting the stage for the subsequent steps of regeneration, which include the infiltration of monocytes and macrophages and myogenesis, i.e., the de novo formation of myofibers. In the absence of platelets or when platelets lack CXCL7 (CXCL7KO), the recruitment of neutrophils to injured muscles is defective. This in turn leads to unresolved tissue damage, excessive recruitment of macrophages at later phases of regeneration, and to high levels of atrophic ligands that stunt the growth of newly-formed myofibers. Neo-angiogenesis is also reduced. Consequently, post-injury muscles arising from regeneration in the absence of early-stage platelet-initiated chemokine signaling display reduced myofiber size and lower muscle force production. Similar results are found with the experimental depletion of neutrophils. Altogether, these findings indicate a key role for platelet-induced chemokine signaling in ensuring optimal muscle regeneration by guiding the recruitment of neutrophils to injured muscles in the early phase after injury.

그림 8: 혈소판은 혈소판에서 분비되는 케모카인 CXCL7을 통해 손상된 근육으로 호중구의 모집을 유도함으로써 골격근 재생을 촉진한다.

손상에 반응하여 혈소판은 골격근에 국소화되어 혈전을 형성하며, 호중구 화학유인물질인 혈소판 특이적 케모카인(예: CXCL7)의 분비를 통해 호중구의 모집을 촉진한다. 호중구 침윤은 세포 잔해물을 제거하고 단핵구 및 대식세포의 침윤과 근육 형성(즉, 근섬유의 신규 형성)을 포함한 재생의 후속 단계를 위한 기반을 마련함으로써 근육 수리를 촉진하는 것으로 알려져 있다. 혈소판이 없거나 혈소판이 CXCL7을 결핍하는 경우(CXCL7KO), 손상된 근육으로의 호중구 모집이 결핍된다. 이는 결국 해결되지 않은 조직 손상, 재생 후기 단계에서의 과도한 대식세포 모집, 그리고 새로 형성된 근섬유의 성장을 저해하는 높은 수준의 위축성 리간드 수준으로 이어집니다. 신생혈관 형성 또한 감소합니다. 결과적으로, 초기 단계 혈소판에 의해 시작된 케모카인 신호 전달이 없는 상태에서 재생된 손상 후 근육은 감소된 근섬유 크기와 낮은 근력 생산을 보입니다. 실험적으로 호중구를 고갈시킨 경우에도 유사한 결과가 관찰된다. 종합하면, 이러한 연구 결과들은 손상 초기 단계에 손상된 근육으로 호중구 유입을 유도함으로써 최적의 근육 재생을 보장하는 데 혈소판 유도 케모카인 신호전달이 핵심적인 역할을 한다는 점을 시사한다.

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Methods

Mouse husbandry

All mice were housed in the Animal Resource Center at St. Jude Children’s Research Hospital, fed a standard chow diet, and handled in accordance with protocols approved by the St. Jude Children’s Research Hospital Institutional Animal Care and Use Committee (IACUC). Additional accreditation of the Animal Resource Center at St. Jude Children’s Research Hospital was provided by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Mice were housed in a ventilated rodent-housing system with a controlled temperature (22–23 °C), 40% humidity, 12-h light/dark cycle, and given free access to food and water. Humane endpoints were not exceeded in any experiment. Euthanasia was performed with carbon dioxide in agreement with the recommendations of the Panel on Euthanasia of the American Veterinary Medical Association.

Mouse models

C57BL6/J male mice (The Jackson Laboratory, JAX#000664) were utilized at 5–6 months of age, a timepoint at which postnatal skeletal muscle growth has halted. CXCL7KO mice were previously described99: also in this case, male mice were utilized at 5–6 months of age. Littermate wild-type males were used as controls in experiments with CXCL7KO mice, which were genotyped before experimental use by Transnetyx. BALB/c male mice (The Jackson Laboratory, JAX#000651) were utilized at 3 months of age for experiments with neutrophil-depleting antibodies and the corresponding IgG2a/b control antibodies.

Antibody-mediated platelet depletion

For platelet depletion experiments, mice were injected via the tail vein with a single dose (100 µg per mouse) of a platelet-depleting antibody (anti-GP1bα; R300, Emfret) or IgG control (R301, Emfret) 2 h before CTX injection into the TA muscle. Platelet-depletion with this anti-GP1bα antibody leads to a greater than 95% reduction in platelet counts, which is achieved within 60 min of antibody injection71,72,73,74. At day 4 from CTX injection, the mice were injected via the tail vein with a second dose of platelet-depleting antibody or IgG control.

Antibody-mediated neutrophil depletion

For neutrophil depletion, mice were injected via the tail vein with 200 µg per mouse of a neutrophil-depleting antibody (anti-Gr1; BioLegend, clone RB6-8C5) or the isotype control (IgG2b; BioXCell, clone LTF-2) 24 h before glycerol-mediated injury of the TA muscle. Subsequently, these mice were further injected by i.p with another neutrophil-depleting antibody (anti-Ly6G; BioXCell, clone 1A8, 200 µg per mouse) or the corresponding isotype control (IgG2a; clone 2A3, 200 µg per mouse) 48 h and 96 h after the first dose of neutrophil-depleting antibody. BALB/c male mice (The Jackson Laboratory, JAX#000651) at 3 months of age were utilized for these experiments because anti-Gr1 and anti-Ly6G antibodies work better in this strain to deplete neutrophils73,135,136,137.

Skeletal muscle injury protocols

For skeletal muscle injury with cardiotoxin (CTX), 50 µL of CTX (cardiotoxin from Naja mossambica mossambica, Sigma cat. no. C9759) was injected into the tibialis anterior (TA) muscle at a concentration of 0.3 mg/mL in PBS whereas the TA in the contralateral leg was mock-injected with PBS. For skeletal muscle injury with glycerol, 70 µL of 50% glycerol (Sigma, cat. no. G5516) was injected into the tibialis anterior muscle whereas the TA in the contralateral leg was mock-injected with PBS. Subsequently, the TA muscles were excised and frozen in isopentane cooled with liquid nitrogen for histology after 1, 7, and 14 days from injury, and snap-frozen in liquid nitrogen for the preparation of tissue homogenates for cytokine arrays.

Immunostaining on tibialis anterior skeletal muscle sections

For immunostaining, TA muscles were bisected at the mid-belly, mounted onto tragacanth gum, and frozen in liquid nitrogen-cooled isopentane (Sigma-Aldrich, cat. no. 277258); 10-µm sections were cut on a cryostat and immunostained as previously done138,139,140,141. Unfixed slides holding the sections were incubated with blocking buffer (PBS with 0.1% Triton X-100, 1% BSA, and 2% horse serum) for 1 h before incubation with primary antibodies, which were all used at 1:150 for immunostaining.

For immunostaining muscle-infiltrating immune cells, the following antibodies were used: anti-MMP9 (R&D Systems, cat. no. AF909) and anti-Ly6G (BioLegend, cat. no. 17-9668-82) to detect neutrophils; anti-F4/80 (BioLegend, cat. no. 123119) to immunostain total macrophages, anti-CD68 to immunostain M1 macrophages (Abcam, cat. no. ab125212), and anti-CD206 (Macrophage Mannose Receptor; 6068c2, Biolegend, cat. no. 141711) to immunostain M2 macrophages; and anti-GP1bβ antibodies to detect platelets (Emfret, cat. no. X649). In addition, rat anti-laminin α2 antibodies (4H8-2; Santa Cruz, cat. no. sc-59854) or WGA (Wheat Germ Agglutinin, Alexa Fluor 555 conjugate, ThermoFisher, cat. no. W32464) were used to delineate the myofiber boundaries. Anti-eMHC antibodies were used to detect embryonic myosin heavy chain (anti-MYH3, Santa Cruz, cat. no. SC-5309). Anti-PECAM-1 antibodies (MEC13.3; BD Biosciences) were utilized to identify blood vessels. Immunostaining for Perilipin-1 (Cell Signaling Technologies, cat. no. 9349) was used to identify fat infiltration in skeletal muscles after regeneration81. Nuclei were detected with DAPI (4′,6-diamidino-2-phenylindole; Roche, cat. no. 10236276001). For the analysis of immune cell infiltration, the images were threshold-adjusted and the percentage of the muscle field area occupied by immune cells was calculated by using the Nikon Elements software (version 4.11.0). All images within an experiment were processed equally.

For myofiber size and type analysis, TA muscle sections were incubated with antibodies against type 2 A (DSHB, cat. no. SC-71) and 2B myosin heavy chain (DSHB, cat. no. BF-F3) and rat anti-laminin α2 (4H8-2; Santa Cruz, cat. no. sc-59854) overnight at 4 °C. The sections were then washed and incubated with secondary antibodies for type 2A (anti-mouse IgG1 Alexa488, Life Technologies cat. no. A21121), type 2B (anti-mouse IgM Alexa555, Life Technologies cat. no. A21426), and laminin (anti-rat IgG Alexa647, Life Technologies cat. no. A21247). The whole tibialis anterior section was imaged on a Nikon C2 confocal microscope with a ×10 objective and stitched to compile an overview of the muscle. The myofiber types and sizes were analyzed with the Nikon Elements software (version 4.11.0) by using the inverse threshold of laminin α2 staining to determine myofiber boundaries. The myosin heavy chain staining was used to classify type 2B myofibers (red), type 2A (green), and presumed 2× myofibers (black) that were not stained for 2B or 2A. After the myofibers were classified and the parameters measured, the Feret’s minimal diameter was used as measurement of the myofiber size due to its accuracy in estimating the size of unevenly shaped or cut objects.

For the quantification of the number of myofibers, all myofibers in the cross-sections of entire tibialis anterior muscles were counted based on the myofiber borders identified by laminin immunostaining. The size and number of myofibers were measured from the inverse images of laminin immunostaining (for identifying myofiber borders), excluding myofibers with diameters <2 and >100 µm. To categorize myofiber types, the intersections of the inverse images of laminin and myosin heavy chain-specific staining were used. These analyses were performed using the Nikon Elements software (version 4.11.0) and the “Object count” function.

Hematoxylin and eosin (H&E) staining

Frozen TA muscle sections (10 μm-thick) were mounted on positively charged glass slides (Superfrost Plus; Thermo Fisher Scientific, Waltham, MA), and dried at room temperature for 1 h. Tissue sections were then stained with H&E according to standard procedures142.

Schemes

Schemes were drawn with BioRender.

Muscle force measurements

The measurement of the twitch and tetanic force of the tibialis anterior (TA) muscle was done as previously described139,140,143 and normalized by the TA mass. Mice were deeply anesthetized via isofluorane and monitored throughout the experiment. The distal tendon of the tibialis anterior was carefully dissected and individually tied with braided surgical silk (CynaMed Suture Thread with Needle; 12, 5/0, 19 mm Blade, 1/2 Reverse Cutting). The sciatic nerve was exposed and all branches were cut except for the common peroneal nerve. The foot was secured to a platform and the knee immobilized using a stainless-steel pin. The body temperature was monitored and maintained at 37 °C. The suture from the tendon was individually attached to the lever arm of a 305B dual-mode servomotor transducer (Aurora Scientific, Ontario, Canada). Muscle contractions were then elicited by stimulating the distal part of the sciatic nerve via bipolar electrodes, using supramaximal square-wave pulses of 0.2 msec (701 A stimulator; Aurora Scientific). Data acquisition and control of the servomotor were conducted using a Lab-View-based DMC program (version 5.202; Aurora Scientific). Optimal muscle length (Lo) was determined by incrementally stretching the muscle until the maximum isometric twitch force was achieved. The fatigue resistance protocol consisted of 60 tetanic contractions (60 Hz stimulation/500-ms duration) every 3 s for a total of 3 min.

Automated image analysis

H&E slides were scanned at 20x. For estimating the infiltration of immune cells into skeletal muscles, the Ilastik machine learning software144 was used to segment the regions with immune infiltration and the total muscle tissue area in each slide, which lead to quantify the ratio of immune infiltration versus the total muscle area. For quantifying myofibers with centrally-located nuclei, the StarDist145 deep learning model was used to segment the nuclei in each slide. Subsequently, the Ilastik software package was used to generate a stack of 36 features for each slide. We used the feature stacks and the segmented nuclei as an input for the Ilastik object classification workflow and trained a classifier to detect centrally located nuclei. The muscle segmentation and immune infiltration mask was used to remove the nuclei located outside the muscle tissue and the nuclei located in the immune-infiltrated muscle regions.

In the graphs that report these quantifications, the center line is the median whereas the lower and upper hinges correspond to the first and third quartiles (the 25th and 75th percentiles). The upper whisker extends from the hinge to the largest value, no further than 1.5 * IQR (inter-quartile range) from the hinge. The lower whisker extends from the hinge to the smallest value, at most 1.5 * IQR from the hinge. The statistical analysis was done with the two-way Mann–Whitney U statistical test, which was run in R with the Wilcox function.

ELISA assays to quantify the levels of chemokines in the mouse plasma

ELISA assays were done according to manufacturer instructions by using the mouse CXCL1/KC quantikine ELISA kit (R&D, cat. no. MKC00B), the mouse CXCL4/PF4 quantikine ELISA kit (R&D, cat. no. MCX400), the mouse CXCL5/LIX quantikine ELISA kit (R&D, cat. no. MX000), and the RayBio mouse CXCL7/TCK-1 ELISA Kit (RayBiotech, cat. no. ELM-TCK1-1).

ELISA assays to quantify the levels of total (active and inactive) versus inactive CXCL7 in injured and uninjured muscles

Total CXCL7 (active and inactive) was quantified from lysates using the RayBio mouse CXCL7/TCK-1 ELISA Kit (RayBiotech, cat. no. ELM-TCK1). Quantification of the uncleaved, inactive form of CXCL7 was performed with a modified version of this kit by using a PPBP polyclonal antibody specific for inactive CXCL7 (Invitrogen, cat. no. PA5-115070, 1:300) as the primary detection antibody and an anti-rabbit HRP-linked secondary antibody (Cell Signaling, cat. no. 7074, 1:3000).

Neutrophil chemotaxis assays

In vitro chemotaxis assays were done as previously described73. Mouse blood was collected by cardiac puncture and red blood cells were lysed for 5 min in lysis buffer (155 mM NH4Cl, 12 mM NaHCO3, 0.1 mM EDTA). The leukocytes were then washed in FACS buffer, blocked with CD16/32 antibody (BioLegend), and stained with fluorophore-conjugated primary antibodies (anti-mouse CD11b and Ly6G). Different recombinant chemokines (rCXCL4, rCXCL5, and rCXCL7; R&D cat. no. 595-P4, 433-MC, and 1091-CK) were added as chemoattractants at 2 µg/mL into the lower chamber of a ChemoTx chemotaxis system (Transwell filter with 5-µm pore size; Neuroprobe). Stained leukocytes were plated in the upper chamber. Both the upper and lower chambers contained RPMI. After 2 h, the content of the lower chamber was collected and mixed with propidium iodide (for assessing cell viability) and Bright Count absolute counting beads (Invitrogen). Samples were then analyzed by FACS to determine the number of migrated CD11b+Ly6G+ neutrophils.

Cytokine antibody arrays

TA muscle tissues were homogenized in a bullet blender at 4 °C with 0.5-mm zirconium beads and RayBio Lysis Buffer for antibody arrays (RayBio Lysis Buffer; AA-LYS-10 mL) with protease inhibitors. After homogenization, the lysates were centrifuged for 5 min at 10,000 x g to remove tissue debris and the supernatant was collected and used for probing the cytokine arrays. 10 µL of the supernatant was used for protein quantitation. For each sample, 350 µL (at ~1–2 mg/mL) were applied to the Quantibody Mouse Cytokine Antibody Array 640 (RayBiotech, catalog #: QAM-CAA-640), a combination of 16 non-overlapping antibody arrays to quantitatively measure 640 mouse cytokines, and processed by the manufacturer according to the standard procedures listed in the manual for this product. The final concentration of each target cytokine (pg/mL) in each sample was utilized for hierarchical clustering and to generate a heatmap. Specifically, the cytokine heatmap was generated from z-scores of cytokine protein levels, after assigning a base value to each cytokine using 2 × z-score (min non-zero), which was used to replace missing values, i.e., concentrations of 0 pg/mL. Subsequently, a clustering method of UGPMA (unweighted pair group method with arithmetic mean) and similarity measure of correlation were applied, using the Spotfire (v7.5.0, TIBCO) Hierarchical Clustering tool.

Statistics and reproducibility

Data organization, scientific graphing, and statistical analyses were done with Microsoft Excel (version 14.7.3) and GraphPad Prism (version 8). The unpaired two-tailed Student’s t test was used to compare the means of two independent groups to each other. One-way ANOVA with Tukey post hoc testing was used for multiple comparisons of more than two groups of normally distributed data. Two-way ANOVA with post hoc testing (typically Tukey for multiple comparisons between groups, and Sidak for comparisons within a group) was used for multiple comparisons of more than two groups of normally distributed data in presence of two independent variables. The n for each experiment can be found in the figure legends and represents independently generated samples (e.g., TA muscles) sourced from distinct mice. Bar graphs display the mean ± SD. A significant result was defined as P < 0.05. Throughout the figures, asterisks and ampersand symbols indicate the significance of P values: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Ampersand values refer to the comparison of muscles from control versus platelet-depleted or versus Cxcl7ko mice at a given timepoint of regeneration. Representative micrographs are derived from the analysis of multiple muscles.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

All the primary data corresponding to the figures and supplementary figures of this study are available in the Source data File. Source data are provided with this paper.

References

1.  
2.  
3.  
4.  
5.  
6.  
7.  
8.  
9.